JP2018535684A - Use of microRNA precursors as drugs to induce proliferation of CD34 positive adult stem cells - Google Patents

Abstract

The present invention generally relates to compositions for inducing proliferation and / or regeneration of CD34 positive adult stem cell populations that can be used in the development of drugs / vaccines and / or therapies for various degenerative diseases related to human aging, and Regarding the method. The present invention also teaches the production and purification methods necessary for the production of high quality, large quantities of small hairpin RNA (shRNA) compositions. These compositions, such as microRNA precursors (pri-miRNA and pre-miRNA) and small interfering RNAs (siRNA) can be used for the treatment of human aging related diseases. Examples of human aging-related diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, osteoporosis, diabetes and cancer. The resulting shRNA, preferably pre-miRNAs, can be used in the development of therapeutic drugs for human degenerative diseases, specifically by a mechanism that induces CD34 positive stem cell proliferation and / or regeneration.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This invention is filed on Dec. 2, 2015 and has priority to U.S. Provisional Application No. 62 / 262,280 entitled “miR-302 Attenuates Aβ-induced Neurotoxicity through Activation of Akt Signaling”. Insist. The present invention was also filed on February 19, 2016, and has the priority of U.S. Patent Application No. 15 / 048,964, whose title is `` A Composition and Method of Using miR-302 Precursors as Drugs for Treating Alzheimer's Diseases ''. Insist. Further, the present invention claims the priority of US Patent Application No. 15 / 167,226 filed on May 27, 2016 and titled “Use of MicroRNA Precursors as Drugs for Inducing CD34-positive Adult Stem Cell Expansion”. doing.

  The present invention relates generally to compositions and methods for inducing proliferation and / or regeneration of CD34 cells with small hairpin RNA (shRNA) molecules, preferably microRNA precursors (pre-miRNA) and / or siRNA. These molecules can be used in the development of drugs / vaccines and / or therapies for a variety of degenerative diseases related to human aging. The present invention also teaches the production and purification methods necessary for the production of high quality, large quantities of small hairpin RNA (shRNA) compositions. These compositions, such as microRNA precursors (pri-miRNA and / or pre-miRNA) and small interfering RNAs (siRNA) can be used for the treatment of human aging-related diseases, such as Alzheimer's Examples include, but are not limited to, Alzheimer's disease, Parkinson's disease, osteoporosis, diabetes and cancer. The resulting shRNAs, preferably pre-miRNAs and siRNAs, can be used in the development of therapeutic drugs for human degenerative diseases, specifically by mechanisms that induce CD34 positive stem cell proliferation and / or regeneration. . In addition, the present invention provides a pre-miRNA having a mechanism called “Cancer Reversion” that can reprogram the malignant characteristics of advanced liver cancer to low benign and thus to a relatively normal stage. Novel drug compositions based thereon are also disclosed. Since cancer reversal is a new concept in drug design, the present invention designs first line drugs for use in cancer therapy through such a novel mechanism.

  Stem cells, like treasure chests with large amounts of active ingredients, stimulate new cell growth / tissue regeneration, repair / recovery of damaged / aged tissue, treatment of degenerative diseases, and tumor / cancer formation / progression. Can be used for prevention. Therefore, the inventors can conceive that these stem cells can be used as a tool for screening, identification and production of novel drugs. Thus, the drugs thus obtained can be used in the development of pharmaceutical and therapeutic applications, for example in the biomedical use of research, diagnosis and / or therapy, devices and / or equipment and combinations thereof.

  MicroRNA (miRNA) is one of the main active ingredients in human embryonic stem cells (hESCs). The types of hESC-specific miRNAs primarily include, but are not limited to, miR-302 family, miR-371-373 family and miR-520 family members. Among them, the miR-302 family has already been found to have a functional effect in tumor suppression (Lin et al., 2008 and 2010). MiR-302 contains eight (8) family members, four (4) sense miR-302 (a, b, c and d) and four (4) antisense miR-302 * (a *, B *, c * and d *). These sense and antisense members are partially matched and can each form a double-stranded body. The precursors of miR-302 are miR-302a and a * (pre-miR-302a), miR-302b and b * (pre-miR-302b), miR-302c and c * (miR-302c) and miR, respectively. -302d and d * (pre-miR-302d), with a binding sequence (stem loop) at one end. In order to activate miR-302 functions, the miR-302 precursor (pre-miR-302) is first transformed into mature miR-302s by treatment with cellular ribonuclease III Dicers (RNaseIII Dicers), and then an algo Forms RNA-induced silencing complexes (RISCs) with argonaute protein, and then causes RNA interference (RNAi) -directed degradation or translational repression of target gene transcripts (mRNAs), especially oncogenic gene mRNAs ( Lin et al., 2008, 2010 and 2011).

  MiR-302 is the most abundant ncRNA type found in hESCs and induced pluripotent stem cells (iPSCs). In our previous studies, ectopic overexpression of miR-302, which is higher than the level in hESCs, indicates that the relatively slow cell cycle rate (20-24 hours) of early human fertilized eggs, similar to the morula stage Showed that both normal human cells and cancer cells can be reprogrammed to hESC-like iPSCs (Lin et al., 2008, 2010 and 2011; EP 2198025; US 12 / 149,725; US 12 / 318,806; US 12 / 792,413). Relative quiescence is a distinct feature of these miR-302-derived iPSCs, but the four-factor induction (Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog in hESCs and other previous reports) -Lin28) iPSCs all exhibit a high proliferative cell cycle rate similar to tumor / cancer cells (12-15 hours / cycle) (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007) Wang et al., 2008). To disclose such tumor suppressive effects of miR-302, the inventors identified for two miR-302 target G1-checkpoint modulators, including cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2010; US 12 / 792,413; US 13 / 964,705). Cell cycle progression is driven by activation of cyclin-dependent kinases (CDKs), positive regulatory subunits cyclins and negative regulators CDK inhibitors (CKIs such as p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1 and p27Kip1) are known to form functional complexes. In mammals, different cyclin-CDK complexes are involved in the regulation of transitions at different times in the cell cycle, for example, cyclin-D-CDK4 / 6 is used for G1 phase progression, cyclin-E-CDK2 is Cyclin-A-CDK2 is used for S-phase progression, and cyclin-A / B-CDC2 (cyclin-A / B-CDK1) is used for M-phase entry. Therefore, as is clear from the inventors' research, the tumor suppressor function of miR-302 is caused by co-suppression of the cyclin-E-CDK2 and cyclin-D-CDK4 / 6 pathways during the transition period from G1 to S.

  miR-302 can be used in the design and development of new anti-cancer drugs / vaccines, but its production is problematic. Natural miR-302 is found only in human pluripotent stem cells such as hESCs, and its resources are extremely limited. Alternatively, synthetic small interfering RNA (siRNA) can be used to mimic pre-miR-302. However, because the pre-miR-302 structure is formed by two mismatched miR-302 and miR-302 * strands, such a perfectly matched siRNA mimic can replace the function of miR-302 *. The sequence is completely different from the antisense strand of siRNA. For example, the antisense strand of siRNA-302a mimic is 5'-UCACCAAAAC AUGGAAGCAC UUA-3 '(SEQ.ID.NO.1), while natural miR-302a * is 5'-ACUUAAACGU GGAUGUACUU GCU-3' (SEQ.ID.NO.2). Since miR-302 functions are generated by both its sense miR-302 and antisense miR-302 * strands, previous reports using such siRNA mimics show results that differ from those of natural miR-302. It was. On the other hand, the inventors' recent research on iPSCs can provide an alternative solution for the production of pre-miR-302 (EP 2198025; U.S. 12 / 149,725; U.S. 12 / 318,806). However, the cost of growing such iPSCs is still too high to be used for industrial manufacturing.

  Alternatively, prokaryotic competent cells may be used as a method capable of producing human microRNAs and precursors thereof. However, prokaryotic cells do not have some essential enzymes necessary for the expression and processing of eukaryotic microRNAs such as Drosha and Dicer. Prokaryotic RNA polymerase cannot efficiently transcribe small RNAs having highly secondary structures, such as hairpin pre-miRNAs and shRNAs. In fact, the bacterial genome does not encode true microRNA and bacteria do not naturally express microRNA. Therefore, if the inventor can express human microRNAs in prokaryotes, the resulting microRNAs are pri-miRNA (a large primary cluster of multiple pre-miRNAs) and / or pre-miRNA ( It is most likely to retain a precursor structure morphology similar to one single hairpin RNA). All the above problems exist, but the real challenge is how to express human microRNAs in prokaryotes. In order to overcome this main problem, the inventor's priority application, US 13 / 572,263, has already established a first step method. However, it is not yet clear whether microRNAs (pro-miRNA) produced by these prokaryotes have the same structure and function as their human counterparts. Also, the pro-miRNAs thus obtained are not suitable for direct application to therapy because they can be contaminated with bacterial endotoxins.

  As can be seen from the current textbooks, it is well known that those having ordinary knowledge in the technical field to which the present invention belongs have different prokaryotic and eukaryotic transcription mechanisms and are therefore incompatible with each other. For example, based on current understanding, eukaryotic RNA polymerase does not bind directly to the promoter sequence, and prokaryotic RNA polymerase, whereas additional co-proteins are required to initiate transcription. Forms a holoenzyme that initiates transcription by binding directly to a promoter sequence. Normally, eukaryotic messenger RNA (mRNA) is synthesized in the cell nucleus by RNA polymerase II (pol-2), then processed and output to the cytoplasm and used for protein synthesis. Translation is also known to occur simultaneously with the same part of DNA at the same location. This is because prokaryotes, such as bacteria and archaea, do not have any cell nucleus-like structure. Therefore, due to these differences, it is difficult and thus impossible for prokaryotic cells to produce eukaryotic RNAs using eukaryotic promoters.

  In the prior art, attempts have been made to produce mammalian peptides and / or proteins in bacterial cells. For example, Buechler US Pat. No. 7,959,926 and Mehta US Pat. No. 7,968,311 use bacterial or phage promoters. To initiate expression, the desired gene is cloned into a plasmid vector driven by a bacterial or phage promoter. The gene must not contain any non-coding introns, since bacteria do not have any RNA splicing mechanism to process introns. Then, by introducing the vector thus obtained into competent strains of bacterial cells such as Escherichia coli (E. coli), gene transcripts (mRNAs) are expressed and mRNAs are further translated into proteins. . However, bacterial and phage promoters such as the Tac, Lac, T3, T7 and SP6 RNA promoters are not pol-2 promoters, and their tendency for transcriptional activity is a false trending process that causes mutations. In addition, Mehta further teaches that glycerol / glycerin can be used to improve bacterial transformation efficiency, but RNA transcription, especially prokaryotic RNA driven by pol-2 promoter There is no teaching about enhancing transcription. Because there is no possible compatibility between eukaryotic and prokaryotic transcription systems, such prior art is still limited to gene expression using prokaryotic RNA promoters in prokaryotes.

  Due to system incompatibility and possible endotoxin contamination problems, there is traditionally no way to produce human pre-miRNA / shRNA class drugs in prokaryotes. Also, pre-miRNA / shRNA is about 70-85 nucleotides in length, too large and too expensive to manufacture due to RNA synthesis mechanisms. In order to overcome these problems, the present invention provides the following new breakthroughs: by adding several distinct chemicals that mimic several transcriptional cofactors, Create a new adaptive environment for prokaryotic cells without using the wrong prokaryotic promoter and use eukaryotic pol-2 and / or pol-2 family promoters to transcribe desired pre-miRNAs and shRNAs be able to. The advantages of this method are as follows. First, the growth of bacteria is fast and can be produced economically and in large quantities. Second, it is easy to treat because there is no need to grow dedicated hESCs or iPSCs. Third, it has high fidelity productivity for RNA transcription driven by the pol-2 promoter. Fourth, since prokaryotes lack true microRNA, they have the desired purity of microRNAs. Finally, since there is no endotoxin, it can be further removed by several chemical treatments. Therefore, a method of producing human pre-miRNAs and / or shRNAs in prokaryotic cells without the problem of system incompatibility and endotoxin contamination is highly desirable. In addition, the drug thus obtained can exhibit a novel therapeutic effect other than the currently known function of microRNA.

European Patent Application No. 2198025 US Pat. No. 7,959,926 US Pat. No. 7,968,311

Lin SL, Chang D, Chang-Lin S, Lin CH, Wu DTS, Chen DT and Ying SY. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA 14, 2115-2124 Lin SL and Ying SY. (2008) Role of mir-302 microRNA family in stem cell pluripotency and renewal.Ying SY. (Ed.) Current Perspectives in MicroRNAs.Springer Publishers press, New York, pp. 167-185 Lin SL, Chang D, Ying SY, Leu D and Wu DTS. (2010) MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 / 6 cell cycle pathways.Cancer Res. 70, 9473- 9482 Lin SL, Chang D, Lin CH, Ying SY, Leu D and Wu DTS. (2011) Regulation of somatic cell reprogramming through inducible mir-302 expression.Nucleic Acids Res. 39, 1054-1065 (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 126, 663--676 Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 Wernig et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 Wang et al. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478-1483

  The principles of the present invention are based on the property of differences and incompatibility between prokaryotic and eukaryotic RNA transcription systems. Naturally, prokaryotic RNA polymerases do not recognize eukaryotic promoters and vice versa. However, the present invention has identified chemical agents that induce and / or enhance RNA transcription driven by eukaryotic promoters in prokaryotes as transcription inducers. Thus, the knowledge taught in the present invention is a new breakthrough beyond the perception of the difference between current prokaryotic and eukaryotic transcription systems.

  The present invention relates to inducible gene expression compositions that stimulate and / or enhance RNA transcription driven by eukaryotic promoters in prokaryotes by several chemical inducers. These chemical inducers have not yet been used in cell culture media due to their antibacterial and / or bactericidal properties, and 3-morpholinopropane-1-sulfonic acid [or 3- (N-morpholino) propanesulfone Acid; referred to as MOPS], glycerin and ethanol, and functional analogues thereof, such as 2- (N-morpholino) ethanesulfonic acid (MES), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) And mannitol. It can be imagined that chemicals with similar structures such as these transcription inducers can share similar functions. For example, MOPS is usually used as a buffer in the degradation of bacterial cells and is therefore not suitable for growing bacteria. On the other hand, ethanol is a well-known disinfectant, and glycerin is usually used to transform bacteria by destabilizing the cell walls of bacteria. Glycerin is antibacterial and ethanol is bactericidal. Each is shown. In view of the above-mentioned known functions of MOPS, ethanol and glycerin, those who have ordinary knowledge in the technical field to which the present invention pertains, if they do not know the knowledge of the present invention beforehand, It is unexpected to induce gene expression driven by eukaryotic promoters in prokaryotic cells using the above chemicals / volume concentration).

  Based on the above knowledge, the present invention is a design and method for producing human microRNA precursors (pre-miRNAs) and / or shRNAs by prokaryotic cells as therapeutic drugs and / or vaccines for cancer therapy. More specifically, the present invention is a design and method for producing special pre-miRNA class drugs (called pro-miRNAs) from prokaryotic cells, which drugs are malignant characteristics of advanced human cancer cells. Can be reprogrammed to a low degree of benign, and thus relatively normal. These pro-miRNAs are tumor suppressor microRNAs (TS-miRNAs), miR-302a, b, c, d, e and / or f precursors (pre-miR-302s) and their natural family clusters and their It is preferred to resemble homologues / derivatives and / or combinations of artificially redesigned small hairpin RNAs (shRNAs). Homologue / derivative design of pro-miRNA-like shRNA includes imperfect and perfect matching hairpin compositions of pro-miRNA and its homologous small interfering RNA (siRNA), single unit or multiple unit clusters Can be formed. These designs can improve target specificity and / or reduce the pro-miR-302 copy number required for effective delivery and therapy. Human cells applied to such drug treatment include in vitro, in vitro and / or in vivo normal cells, tumor cells and cancer cells.

  The prokaryotic cell used in the present invention is a bacterial competent cell, particularly E. coli, and the chemical inducer is preferably MOPS, ethanol, glycerin or a mixture thereof. The eukaryotic RNA promoter used may also be a eukaryotic pol-2 promoter (ie EF1α promoter) or a pol-2 compatible (pol-2 family) viral promoter (ie cytomegaloviral CMV promoter). preferable. The eukaryotic RNA promoter-mediated gene is selected from the group consisting of microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA), precursors and homologues thereof, and combinations thereof It can encode non-coding and / or protein-encoding RNA (eg, gene transcripts containing introns). To induce gene expression, transfect prokaryotic cells with eukaryotic RNA promoter-mediated genes and grow for> 24 hours in medium similar to Luria-Bertani (LB) medium at 37 ° C with the addition of chemical inducers Let

  In order to demonstrate that these chemical inducers can induce the production of human microRNA in prokaryotes, the inventors have invented from the inventors' priority applications U.S. Patent Application Nos. 12 / 149,725 and 12 / 318,806. The lentiviral vector pSpRNAi-RGFP-miR302 is modified with a new plasmid vector pLenti-EF1a-RGFP-miR302, and the gene expression of the SpRNAi-RGFP is driven by a eukaryotic promoter such as EF1α or CMV (FIG. 1A). Then, as shown in FIG. 1B, the inventors transformed Escherichia coli competent cells using it, and then visually observed the production of red fluorescent protein (RGFP) by measuring the transcription rate and process of microRNA miR-302. Used as a mark. As shown in FIG. 5 and FIG. 6, the miR-302 family cluster is also modified so that the 5′-intron region of the RGFP gene [eg, 5′-untranslated region (5′-UTR) or first intron] Since each RGFP mRNA is encoded, one 4 hairpin miR-302 precursor cluster (pri-miR-302) and / or 4 one hairpin miR-302 precursors (pre-miR-302s) Lead until it occurs. Since prokaryotes lack RNase III Dicer, the pri-miR-302 transcript is eventually degraded (by several single-stranded RNases in E. coli) into 1 hairpin pre-miR-302s, both of which are extracted. Furthermore, it can be used as a therapeutic drug in the present invention. Broadly speaking, 5′-UTR and 3′-UTR are considered part of introns in the present invention.

  All miR-302 members share the same sequence 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3) in the previous 5'-17 (17) nucleotides and mature microRNA Contains more than 82% homology at 23 nucleotides in total length. According to the prediction results of the online calculation programs TARGETSCAN (http://www.targetscan.org/) and PICTAR-VERT (http://pictar.mdc-berlin.de/), these miR-302 Targets the same genes simultaneously and includes over 600 human genes. MiR-302 is a target that overlaps with various family members of mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373, mir-374 and mir-520. They also share genes and all can have similar functions. Most of these target genes are developmental signals and transcription factors that are involved in the initiation and / or establishment of several lineage-specific cell differentiation during early embryonic development (Lin et al., 2008). Since many of these target genes are also well-known oncogenic genes, miR-302s is likely to prevent normal hESC growth as a tumor suppressor from biasing to tumor / cancer formation.

Induction of gene expression driven by eukaryotic promoters in prokaryotes E. coli competent cells were transformed into pLenti-EF1α-RGFP- using the z-competent E. coli transformation kit (Zymo Research, Irvine, CA) Luria- transformed with miR302 plasmid (Figure 1A) and supplemented with a mixture of 0.1% (v / v) MOPS and 0.05% (v / v) glycerin (inducing agent) under frequent agitation at 37 ° C and 170 rpm Incubate with Bertani (LB) medium. As shown in Figure 2, after overnight incubation, transformed E. coli competent cells express a highly abundant red RGFP protein, as clearly seen in the color of the LB medium. Blank E. coli do not show RGFP. As evident from the presence of functional RGFP, both the encoding RNA and protein were both successfully generated and processed in competent cells.

  To further demonstrate the specificity of gene expression induced by chemical inducers, two transformed E. coli strains are prepared. One carries the pLVX-Grn-miR302 + 367 plasmid vector containing the green fluorescent protein (GFP) gene driven by the CMV promoter, and the other carries the pLenti-EF1α-RGFP-miR302 vector. . As shown in FIG. 3, after overnight incubation with 0.1% (v / v) MOPS alone, E. coli transformed with pLVX-Grn-miR302 + 367 turned green and pLenti-EF1α-RGFP-miR302 Transformed E. coli still shows a red color. As is clear from this result, chemical inducers such as MOPS can stimulate the transcription of specific RNA and the development of its related proteins via eukaryotic pol-2 or pol-2 class viral promoters. . In particular, it should be noted that the production of RGFP and GFP was so abundant that E. coli cells were even visible as stained with red and green, respectively.

  As shown in FIG. 4, of all the chemicals tested in the present invention, three of the most potent inducers are MOPS, glycerin and ethanol. As shown in FIG. 5 and Example 3, further Western blot analysis demonstrates the quantitative results with induced RGFP. Bacterial RuvB protein normalizes RGFP expression as a housekeeping standard. It has also been found that the inductivity of these inducers identified is dose dependent in proportion to their concentration. In the absence of any treatment, the negative control E. coli cells show only their original color in the absence of any fluorescent stain. Thus, according to all these results, the present invention clearly demonstrates the novel chemically-derived composition and its RNA driven by eukaryotic pol-2 in prokaryotic cells or driven by a pol-2 family virus promoter Provides applications for regulating the occurrence of Based on the above argument, those who have ordinary knowledge in the technical field to which the present invention belongs can use other genes or related cDNAs in place of the RGFP gene to produce functional RNAs and related proteins in prokaryotes. it is obvious.

Induction of microRNA expression driven by eukaryotic promoters in prokaryotes In accordance with the RGFP induction experiments shown above, the inventors have pri- / pre-miR in pLenti-EF1α-RGFP-miR302 transformed cells with or without chemical induction -302s and its mature miR-302s expression is further measured. As shown in FIG. 6 and Example 4, the quantitative results with induced pri- / pre-miR-302 were already demonstrated by Northern blot analysis. The expression of pri- / pre-miR-302 is similar to the result of RGFP induction in Fig. 4 and Fig. 5 and can be greatly detected in transformed cells treated with MOPS, glycerin or ethanol, but not in the blank . This indicates that these chemical inducers stimulate the expression of encoded pri- / pre-miRNAs in prokaryotic cells substantially via the eukaryotic pol-2 promoter (FIG. 6). Due to the structural similarity between pre-miRNAs and shRNAs, those with ordinary knowledge in the technical field to which the present invention belongs can produce other types of pri- / pre-miRNA species using the present invention. Is clear. Examples include, but are not limited to miR-34, miR-146, miR-371-373 and miR-520. For clarity, pri- / pre-miRNAs produced by these prokaryotes are referred to as pro-miRNAs.

  Since pLenti-EF1α-RGFP-miR302 contains the miR-302 family cluster (Figure 1A and Figure 1B) located in the 5'-UTR of the RGFP gene, as shown in Figure 1B, also induced RGFP gene expression The miR-302 cluster (pri-miR-302) and its derivatives pre-miR-302a, b, c and d (pre-miR-302s) are generated. Since prokaryotes lack RNase III Dicer, pri-miR-302 and pre-miR-302s thus obtained can be retained as hairpin microRNA precursors and can be used for the development of therapeutic drugs. It was done. In human cells, these pre-miR-302s and pri-miR-302 can be used to cause their tumor suppressor function by being processed into mature miR-302. Similarly, the present invention can be used to produce other types of TS-miRNA species and precursors thereof. Examples include the miR-34a, miR-146a, miR-373 and miR-520 families.

  The resulting pro-miRNAs can be easily extracted from competent E. coli cells (Examples 5 and 6) and further purified (FIG. 10A and FIG. 10B) by high performance liquid chromatography (HPLC). Within the purified pro-miR-302s, the inventors identified microRNA microarray analysis (Figures 11B and 12) and RNA sequencing [Figures 13A (pri-miR-302) and 13B (pre-miR-302s)] Was used to recognize all miR-302 family members (miR-302a, a *, b, b *, c, c *, d and d *). Specifically, as shown in the sequencing results, both of these pro-miR-302s share exactly the same sequence (FIG. 13B) as their natural pre-miR-302 counterparts. In addition, in order to measure the therapeutic effect on human liver cancer in vivo, the inventors formulated these pro-miR-302s as soluble drugs used for IV / in vivo injection (Example 11). As shown in Figure 14, after 3 injection treatments, the pro-miR-302 drug successfully treated the average size of cancer, reducing the volume of human liver cancer transplanted in vivo more than 90% Reduced to less than 10% for no cancer. In addition, the results of histological examination with hematoxylin & eosin (H & E) staining showed that this remarkable therapeutic effect was not only due to the reported tumor suppressor function of miR-302 (Lin et al., 2010). It was further shown that it depends on another new reprogramming feature that has not yet been discovered. For example, as clearly shown in FIG. 15, the pro-miR-302 drug can be reprogrammed in vivo to a benign stage that approximates the advanced malignant characteristics of human liver cancer almost well to normal liver tissue. These treated cancers can form normal liver-like structures such as classical liver lobules, central veins (CV), and portal triads (PT). Thus, these evidences indicate that pro-miR-302 not only can suppress tumor / cancer cell growth, but also restore malignancy of human cancer to a relatively benign or normal state in vivo, a new cancer drug design Strongly shows that it can lead to therapeutic effects.

  In the present invention, both the plasmid vector and the non-coding RNAs encoded by the plasmid vector (ie, pre-miRNA / shRNA) can be simultaneously amplified in prokaryotic cells, preferably E. coli DH5α competent cells (Examples 1 and 5). And 6). Methods for separating amplified pLenti-EF1α-RGFP-miR302 plasmid DNA and transcribed pri- / pre-miR-302s are described in Examples 5 and 6. The technique of delivering plasmid vectors (ie, pLenti-EF1α-RGFP-miR302) to prokaryotic cells is called cell transformation, and amplified non-coding RNAs (ie, pro- / pri- / pre-miR-302s) Can be delivered to eukaryotic cells by endocytosis, chemical / glycerin injection, peptide / liposome / chemical mediated transfection, electroporation, gene gun penetration, microinjection, transposon It can be selected from the group consisting of / retrotransposon insertion and / or adenovirus / retrovirus / lentivirus infection.

According to the derivatization report of pluripotent stem cells induced by Pre-miR-302 , MiR-302 is demonstrated in US patent application Nos. 12 / 149,725 and 12 / 318,806, the inventor's priority applications. As described, mammalian somatic cells can be reprogrammed into human embryonic stem cells (hESC) -like induced pluripotent stem cells (iPSCs). These iPSCs were used to design and develop many stem cell applications and therapies. However, incubation of these iPSCs and hESCs is extremely costly and time consuming, so collecting miR-302 and its precursors from these pluripotent cells is difficult and less efficient. On the other hand, the production of synthetic shRNA mimics is another possible alternative to the production of pre-miR-302, but the costs are still very high. Also, the similarity between synthetic shRNA and natural pre-miR-302 is well suspected. In order to solve these problems, the present invention provides a simple and cheap method for efficiently producing large quantities of pre-miR-302 in prokaryotes. Also, as shown in FIG. 6 and Example 6 of the present invention, the extraction and purification of pre-miR-302s (pro-miR-302s) produced by these prokaryotes is relatively simple and economical.

  As shown in Examples 5 and 6, the inventor used the Escherichia coli cells transformed with pLenti-EF1α-RGFP-miR302 to produce high quality pLenti-EF1α-RGFP-miR302 vectors and pro-miR-302s. Produced and separated. Both pLenti-EF1α-RGFP-miR302 and pro-miR-302s can be used to generate iPSCs. In accordance with Example 2, when transfecting human skin primary keratinocytes with pro-miR-302s produced in accordance with the present invention, the transfected keratinocytes are reconstituted into hESC-like iPSCs that express the strong hESC mark Oct4. Programmed (Figure 7). In Figure 8 and Example 8, the inventor further performed a sulfite DNA sequencing analysis to ensure that the overall DNA desmethylation is indeed the promoter of both Oct4 and Sox2 genes (two important reprogramming factors). ) And hESC mark. Since global DNA desmethylation and Oct4 expression is known as the first step to reprogram somatic cells to form hESC-like iPSCs (Simonsson and Gurdon, Nat Cell Biol. 6: 984-990, 2004) Pro-miR-302s isolated from an extract of E. coli cells induced by MOPS was demonstrated to be as effective as natural pre-miR-302s that can be used for iPSC induction. Therefore, pro-miR-302 and pre-miR-302 are likely to have the same function in stem cell induction.

Growth and / or regeneration of CD34 positive adult stem cells induced by Pre-miR-302
MicroRNA miR-302 was found to be able to reprogram mammalian somatic cells into embryonic stem cell (ESC) -like induced pluripotent stem cells (iPSCs) (Lin, 2008, 2010, 2011; 12 / 149,725 and 12 / 318,806). These iPSCs have been used to develop a variety of stem cell related applications and therapies to promote modern regenerative medicine. However, miR-302 is found in large quantities only in human ESCs and not in non-differentiated tissue cells. Also, separating miR-302 from human ESCs is very controversial, costly and cumbersome. To solve these problems, the present invention provides hairpin miR-302 molecules and / or precursors / homologs thereof in prokaryotes by providing simple, cheap and rapid inductive compositions and methods. Manufacture a lot of things. Also, as shown in FIG. 6 and Example 6 of the present invention, it is relatively simple and economical to separate miR-302 and / or its precursor from prokaryotic cells.

  As shown in Examples 5 and 6, the inventor used the E. coli cells transformed with pLenti-EF1α-RGFP-miR302 to produce large quantities of high quality pLenti-EF1α-RGFP-miR302 vectors and pre-miR-302s. Produced and separated. Based on the inventors' conventional US patent application Ser. Nos. 12 / 149,725 and 12 / 318,806, it was shown that human ESC-like iPSCs can be generated by using pLenti-EF1α-RGFP-miR302. Moreover, as demonstrated in the inventors' previous research (Lin et al., 2008, 2010 and 2011), the iPSCs thus obtained can be further differentiated into various body tissue cells.

  Application of isolated miR-302 and / or pre-miR-302 molecules can further include induction and proliferation of CD34 positive adult stem cells. As shown in FIG. 17A and FIG. 17B, a relatively low concentration (50-500 μg / mL) as shown in the inventor's recent study of wound healing and cancer therapy using a novel miR-302 combination drug Treatment of isolated miR-302 / pre-miR-302 molecules not only significantly enhances healing of scar-free wounds, but also in vivo CD34 positive adults around wounded areas in pig skin Induces proliferation of stem cells. Based on a comparison of miR-302 treatment (miR-302s / pre-miR-302s + antibiotic ointment) results in Figure 17B and control (antibiotic ointment only) results in Figure 17A, The CD34-positive adult stem cell population (marked with green fluorescent antibody) clearly showed a 40-fold increase. Currently, known types of CD34 positive adult stem cells include, but are not limited to, skin, hair, muscle, blood (hematopoietic), mesenchymal and neural stem cells. Therefore, miR-302 can be used to induce proliferation and / or regeneration of CD34 positive adult stem cells in vivo, so this therapeutic effect is achieved by re-growth and / or regeneration of functional adult stem cells. It can also help treat human degenerative diseases. Examples of degenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, osteoporosis, diabetes and cancer.

Application of pro-miR-302 to in vivo tumor / cancer therapy The inventor's previous study shows that this method is feasible to treat HepG2 cells of human hepatocellular carcinoma in vitro rather than in vivo Was demonstrated (Lin et al., 2010). As shown in FIG. 9, treated tumor / cancer cells are reprogrammed into iPSCs (marked as mirPS-HepG2) to form embryoid body-like cell populations. It was also found that miR-302 can induce more than 95% apoptosis in treated cancer cell populations. The top diagram in FIG. 9 further illustrates flow cytometric analysis where DNA content depends on cell cycle stage, with a significant reduction in mitotic cell populations (from 45.6% to 17.2%) after miR-302 treatment. ) As is apparent from these results, miR-302 can effectively reduce the rapid cell cycle rate of human liver cancer cells, thereby causing significant apoptosis of these cancer cells.

Although the process of cancer progression is considered irreversible due to the accumulation of genetic mutations, the present invention includes a novel pre-reprogrammable in vivo that can reprogram highly malignant cancers to a low benign and thus normal-like stage. -miRNA (pro-miR-302) function is provided, the mechanism of which can be related to the process of natural healing, which is extremely rare and called spontaneous cancer regression. Spontaneous regression of cancer rarely occurs in less than 1 out of 100,000 cancer patients. The inventor has found that pro-miR-302 treatment can increase this rare cure rate to over 90% in human liver cancer. As shown in FIG. 14, the results of treatment of human liver cancer xenografts in SCID-beige nude mice (n = 6) using pro-miR-302s as a drug indicate that the pro-miR-302 drug is a cancer size. Was successfully reduced from 728 ± 328 mm ³ (untreated blank, C) to 75 ± 15 mm ³ (treated with pro-miR-302, T), with an average cancer size of approximately 90 % Reduction indicates that treatment with other synthetic siRNA mimics (siRNA-302) does not provide any similar therapeutic effect.

  Normal liver lobule-like structures (circled and indicated by black arrows), as shown in further histology (Figure 14, rightmost figure), cancer implant treated with pro-miR-302 Observed only in other treatments or controls. This indicates that a reprogramming mechanism has occurred to restore the properties of malignant cancer cells to a relatively normal state (reversal of cancer). This novel reprogramming mechanism is likely to occur due to the gene silencing effect of miR-302 on human oncogenic genes, particularly mutant oncogenic genes involved in cancer progression. By silencing these mutant oncogenic genes, pro-miR-302 can restore the oncogene expression pattern to a normal-like state, thereby producing a therapeutic outcome of cancer reversal. However, since Oct4-positive pluripotent cells are not recognized, it is clear that this in vivo reprogramming mechanism is different from somatic cell reprogramming reported previously (Lin et al., 2008 and 2011). ).

  A more detailed histological study (Figure 15) shows that the pro-miR-302 drug has indeed reprogrammed advanced (IV degree) human liver cancer transplants to a less benign (less than II degree) condition. Further proof. As shown in FIG. 15, the treated cancer implants form classic hepatic lobules that contain central vein (CV) -like and portal triangular (PT) -like structures (indicated by black arrows) and are normal. Very similar to the structure of liver tissue (top figure). Histological comparison between in vivo untreated human liver cancer transplant, human liver cancer treated with siRNA, human liver cancer treated with pro-miR-302 and normal liver tissue (Figure 16) Untreated transplanted human liver cancer (top figure) invasively invades surrounding normal tissues (eg muscle and blood vessels), creating large cell-cell and cancer-tissue fusion structures It also shows that it has formed, demonstrating its advanced malignancy and metastasis. Treatment with siRNA mimic (siRNA-302) does not significantly reduce the malignancy of transplanted liver cancer (middle figure), which is likely due to the short half-life of siRNA in vivo. In contrast, pro-miR-302 treatment not only reprograms the transplanted cancer cells to a normal hepatocyte-like morphology (no fusion) but also successfully penetrates any tissue surrounding the cancer. Suppressed (middle lower figure). Compared with normal liver tissue (bottom diagram), cancer treated with pro-miR-302 has a structure similar to liver lobule, normal glandular cell-like structure, and cell-cell and cancer-tissue association. A very clear border between the sites (black arrows) is clearly shown, indicating that the malignancy of these treated cancers has been greatly reduced to a very benign state. Further treatment with pro-miR-302 drug 6-10 times in succession can completely eliminate cancer xenografts in all 6 samples (n = 6).

A. Definitions For ease of understanding the present invention, some terms are defined as follows.
Nucleotide: A DNA or RNA monomer unit composed of a sugar moiety (pentose), a phosphate group, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety through glycoside carbon (the first carbon of pentose), and the combination of base and sugar is a nucleoside. A nucleoside containing at least one phosphate group and attached to the 3 or 5 end of the pentose becomes a nucleotide. Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is composed of different types of nucleotide units, that is, deoxyribonucleotides and ribonucleotides.

  Oligonucleotide: A molecule composed of 2 or more, preferably more than 3 and generally more than 10 deoxyribonucleic acids (DNAs) and / or ribonucleic acids (RNAs). Oligonucleotides longer than 13 nucleotide monomers are also referred to as polynucleotides. The exact length of the oligonucleotide depends on many factors and is determined by the ultimate function or use of the oligonucleotide. Oligonucleotides can be generated by any method, and this mode includes chemical synthesis, DNA replication, RNA transcription, reverse transcription or combinations thereof.

  Nucleotide analogue: A purine or pyrimidine nucleotide whose structure differs from adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U), but replaces a normal nucleotide in a nucleic acid molecule It is similar enough to be able to.

  Nucleic acid composition: A nucleic acid composition refers to an oligonucleotide or polynucleotide, for example, a DNA or RNA sequence that exhibits a single-stranded or double-stranded molecular structure, or a mixed DNA / RNA sequence.

  Gene: A nucleic acid composition whose oligonucleotide or polynucleotide sequence encodes RNA and / or polypeptide (protein). The gene may be RNA or DNA. The gene can encode non-coding RNA, eg, small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA and its RNA precursors and derivatives. Alternatively, the gene can encode protein-encoding RNA, such as messenger RNA (mRNA) and its RNA precursors and derivatives required for protein / peptide synthesis. Optionally, the gene can encode a protein-encoding RNA that also includes at least one microRNA or shRNA sequence.

  Primary RNA transcript: An RNA sequence that is transcribed directly from a gene without any RNA treatment or modification, and includes mRNA, hnRNA, rRNA, tRNA, snora, snRNA, pre-microRNA, viral RNA and its RNA precursor It can be selected from the group consisting of derivatives.

  Messenger RNA precursor (pre-mRNA): The primary RNA transcript of a protein-coding gene, produced by an intracellular mechanism called transcription in eukaryotes by the eukaryotic RNA polymerase II (Pol-II) mechanism. The pre-mRNA sequence includes a 5 'untranslated region (UTR), 3'-UTR, exons and introns.

  Intron: A part or a plurality of portions of a gene transcription sequence encoding a non-protein reading frame, such as in-frame intron, 5′-UTR and 3′-UTR.

  Exon: A part or parts of a gene transcription sequence (cDNA) that encodes a protein reading frame, including, for example, cDNA used for cellular genes, growth factors, insulin, antibodies and analogs / homologs and derivatives thereof .

  Messenger RNA (mRNA): A collection of pre-mRNA exons, formed after removal by an intracellular RNA splicing mechanism (spliceosome) in an intron and used as a protein-encoding RNA for peptide / protein synthesis. Peptides / proteins encoded by mRNAs include, but are not limited to, enzymes, growth factors, insulin, antibodies and analogs / homologs and derivatives thereof.

Complementary DNA (cDNA): single-stranded or double-stranded DNA that contains a sequence that is complementary to an mRNA sequence and does not contain any intron sequences.
Sense: A nucleic acid molecule that exhibits the same sequence order and composition as a homologous mRNA. The sense structure form is indicated by the sign “+”, “s” or “sense”.

  Antisense: A nucleic acid molecule that is complementary to each mRNA molecule. Antisense structural forms are indicated by the sign “-” or “*”, or are indicated by “a” or “antisense” preceding DNA or RNA, for example, “aDNA” or “aRNA”. It is done.

  Base pair (bp): Pairing of adenine (adenine, A) and thymine (thymine, T) or cytosine (cytosine, C) and guanine (guanine, G) in a double-stranded DNA molecule. In RNA, uracil (U) replaces thymine (T). In general, partnership is realized by hydrogen bonding.

  Base pair (bp): Pairing of adenine (adenine, A) and thymine (thymine, T) or cytosine (cytosine, C) and guanine (guanine, G) in a double-stranded DNA molecule. In RNA, uracil (U) replaces thymine (T). In general, partnership is realized by hydrogen bonding. For example, the sense nucleotide sequence “5′-A-T-C-G-U-3 ′” can form a complete base pair relationship with its antisense sequence “5′-A-C-G-A-T-3 ′”.

  5'-end: the end of a consecutive nucleotide that does not have a nucleotide at the 5 'position, one of which 5'-hydroxy is linked to the next nucleotide 3'-hydroxy by a phosphodiester . Other groups, such as one or more phosphate esters, may be present at the end.

  3'-end: the end of the contiguous nucleotide that does not have a nucleotide at the 3 'position, one of which 5'-hydroxy is linked to the next nucleotide 3'-hydroxy by a phosphodiester . Other functional groups may be present at the terminal, with the hydroxy group being the most common.

  Template: A nucleic acid molecule replicated by a nucleic acid polymerase. The template may be single stranded, double stranded or partially double stranded, depending on the polymerase. The synthesized copy is complementary to at least one strand of the template, duplex or partial duplex template. Both RNA and DNA are synthesized in the 5 'to 3' direction. The two strands of a nucleic acid duplex are always aligned so that the 5 'end of the two strands is at the opposite end of the duplex (which is necessarily the same at the 3' end). ing.

Nucleic acid template: a double-stranded DNA molecule, a double-stranded RNA molecule, or a hybrid molecule, such as a DNA-RNA or RNA-DNA hybrid, or a single-stranded DNA or RNA molecule.
Conserved: When a nucleotide sequence hybridizes non-randomly with the exact complement of a preselected (reference) sequence, the nucleotide sequence is conserved with respect to the preselected sequence Has been.

  Homology or homology: A term indicating the similarity between a polynucleotide and a gene or mRNA sequence. For example, the nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence. Homology may be expressed as a percentage of the total number of nucleotides that are similar.

  Complementary (Complementary or Complementarity or Complementation): Matching base pairs (ie, mRNA and cDNA sequences) that are joined by the base pair rule (“base pair (bp)” rule) and intervene between two polynucleotides. It is a term to indicate. For example, the sequence “5′-A-G-T-3 ′” is complementary to the sequences “5′-A-C-T-3 ′” and “5′-A-C-U-3 ′”. Complementarity may exist between two DNA strands, one DNA and one RNA strand, or two RNA strands. Complementarity may be “partial” or may be “complete” or “total”. Partial complementarity or complementation appears when only a few nucleobases are matched by base pairing rules. Complete or total complementarity or complementation appears when bases are perfectly or perfectly matched between nucleic acid strands. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for detection methods that depend on the amplification reaction and the binding between nucleic acids. Complementarity (percent complementarity or complementation) refers to the ratio of the total number of mismatched bases in a single strand of the nucleic acid. Thus, 50% complementarity means that half of the bases are mismatched and half of the bases are matched. Even if the number of bases of the two strands of nucleic acid is different, the two strands can be complemented. In this case, complementation occurs between a part of the long chain and the short chain, and there is a base in the part of the long chain corresponding to the base paired with the base of the short chain.

Complementary base: A nucleotide that normally pairs when DNA or RNA has a double-stranded structure.
Complementary Nucleotide Sequence: A single nucleotide sequence of a single-stranded DNA or RNA molecule that is sufficiently complementary to the nucleotide sequence of another single-stranded molecule so that it is between two strands. Specifically hybridize through corresponding hydrogen bonds.

  Hybridization (Hybridize and Hybridization): Forms a duplex between nucleotide sequences that are sufficiently complementary and form a complex through base pairing. When a primer (or splicing template) and a target (template) are “hybridized”, these complexes (or hybrids) are sufficiently stable and have a priming function necessary for initiating DNA synthesis by DNA polymerase. provide. Between two complementary polynucleotides, there is a specific, ie non-random, interaction that is competitively inhibited.

  Posttranscriptional Gene Silencing: Knockout or knockdown effect at the stage of mRNA degradation or translational repression of the target gene, usually outer / viral DNA or RNA transgenes Or caused by either small inhibitory RNAs.

  RNA interference (RNAi): a post-transcriptional gene silencing mechanism in eukaryotes, caused by small inhibitory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA) and small interfering RNA (siRNA) . These small RNA molecules usually interfere with the expression of intracellular genes that are fully or partially complementary to small RNAs as gene silencers.

  Gene silencing effect: Cell response after gene function is suppressed, cell cycle attenuation, G0 / G1-checkpoint arrest, tumor suppression, anti-tumor formation (anti -tumorigenecity), apoptosis of cancer cells, and combinations thereof, but are not limited thereto.

  Non-coding RNA: RNA transcript that cannot synthesize peptides or proteins by the intracellular translation mechanism. Non-coding RNA includes long and short regulatory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) and double-stranded RNA (dsRNA). These regulatory RNA molecules usually interfere with the expression of intracellular genes that are fully or partially complementary to the non-coding RNA as gene silencers.

  MicroRNA (miRNA): A single-stranded RNA that can bind to target gene transcripts (mRNAs) that are partially complementary to the sequence of microRNA. The normal length of mature microRNA is about 17-27 oligonucleotides in length, directly degrading intracellular mRNA targets depending on the complementarity between the microRNA and its target mRNA (s), or , Protein translation of the targeted mRNA can be suppressed. Natural microRNAs are found in almost all eukaryotes, serve as defenses against viral infections, and can regulate specific gene expression during plant and animal development. In principle, a single microRNA typically targets multiple target mRNAs to achieve all their functions, while multiple miRNAs target the same gene transcript to enhance gene silencing effects can do.

  MicroRNA precursor (Pre-miRNA): A hairpin-type single-stranded RNA containing stem arms and stem-loop regions that interacts with intracellular RNase III Dicer ribonucleic acid endonuclease to complete the sequence of mature microRNA Or to generate one or more mature microRNAs (miRNAs) that can silence partially complementary target genes or specific groups of target genes. The stem arm of pre-miRNA can form a duplex that hybridizes perfectly (100%) or partially (mismatch), and the stem loop is linked to one end of the stem arm duplex And forms a circular or hairpin-ring structure form necessary for assembly into an RNA-induced silencing complex (RISC) with several Argonaute proteins (AGO).

  Prokaryotic MicroRNA Precursor (Pro-miRNA): Similar to natural microRNA precursor (pre-miRNA), but driven in a prokaryotic competent cell by a eukaryotic promoter and transcribed from an artificial recombinant microRNA expression plasmid Small hairpin RNA. For example, pro-miR-302 is structurally identical to pre-miR-302 (FIGS. 13A and 13B), but in E. coli DH5α competent cells, pLVX-Grn-miR302 + 367 or pLenti-EF1α-RGFP Transcribed from the miR302 vector (Example 1). Prokaryotic cells generally do not express short RNAs with high secondary structure, such as eukaryotic pre-miRNAs, so to produce pro-miRNAs in prokaryotes, pre-miRNAs usually driven by eukaryotic promoters Chemical inducers need to be added to stimulate transcription (Figures 2-4).

  Small interfering RNA (siRNA): A length of approximately 18-27 ribonucleotide duplexes that are perfectly base-paired and can be used to break down transcripts of target genes with almost perfect complementarity Stranded RNA.

  Small or short hairpin RNA (shRNA): A single-stranded RNA comprising a partially or fully matching stem-arm nucleotide sequence that is separated by a pair of unmatched cyclic oligonucleotides to form a hairpin-type structure. A wide variety of natural miRNAs are derived from hairpin RNA precursors, ie, precursor microRNAs (pre-miRNAs).

  Vector: A recombinant nucleic acid composition that can move and stay in different genetic environments, for example, recombinant DNA (rDNA). In general, another nucleic acid is operably linked to it. The vector can self-replicate in the cell, in which case the vector and the segment to be ligated are replicated. A preferred type of vector is an episome, ie, a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are those capable of self-replication and nucleic acid expression. Vectors capable of inducing the expression of genes and / or non-coding RNAs encoding one or more polypeptides are referred to herein as “expression vectors” or “expression competent vectors”. A particularly important vector is the ability to clone cDNA from the generated mRNAs using reverse transcriptase. Vectors include viral promoters and RNA polymerase II (Pol-II or pol-2) promoters or both, Kozak consensus translation initiation site, polyadenylation signals, multiple restriction / cloning sites (Restriction / cloning site), pUC origin of replication, SV40 early promoter to express at least one antibiotic drug resistance gene in replication competent prokaryotic cells, in mammalian cells Selective SV40 origins for replication and / or components from tetracycline responsive elements may be included. The vector structure may be linear or circular single or double stranded DNA, including plasmids, viral vectors, transposons, reverse transposons, DNA transgenes, jumping genes, and their Selected from the group consisting of combinations.

  Promoter: A nucleic acid that is recognized by or bound to a polymerase molecule to initiate RNA transcription. Depending on the purpose of the invention, the promoter may be any sequence capable of initiating synthesis of RNA transcripts by well-known polymerase binding sites, enhancers and analogs, and the required polymerase.

  Eukaryotic promoter: required for transcription of RNA and / or genes, eukaryotic RNA polymerase II (pol-2), pol-2 equivalent and / or pol-2 compatibility (pol-2 class) A sequence of a nucleic acid motif that can be recognized by a viral polymerase to initiate RNA / gene transcription.

  RNA polymerase II (Pol-II or pol-2) promoter: Recognized by eukaryotic RNA polymerase II (Pol-II or pol-2), thereby transcription of eukaryotic messenger RNAs (mRNAs) and / or microRNAs (miRNAs) RNA promoter capable of initiating For example, the pol-2 promoter may be, but is not limited to, a mammalian RNA promoter or a cytomegalovirus (CMV) promoter.

  RNA polymerase II (Pol-II or pol-2) equivalent: selected from the group consisting of mammalian RNA polymerase II (Pol-II or pol-2) and Pol-II compatible (pol-2 class) viral RNA polymerase Eukaryotic transcription mechanism.

  Pol-II compatible (pol-2 class) viral promoter: A viral RNA promoter that can initiate gene and / or RNA expression by a eukaryotic pol-2 or pol-2 equivalent transcription mechanism. For example, the pol-2 family virus promoter may be, but is not limited to, a cytomegalovirus (CMV) promoter or a retroviral long terminal repeat (LTR) promoter.

Cistron: A nucleotide sequence that encodes an amino acid residue sequence and includes upstream and downstream DNA expression control elements in a DNA molecule.
Intron excision: A cellular mechanism that plays a role in RNA processing, maturation and degradation, including RNA splicing, exosome digestion, nonsense mutation-dependent degradation (NMD) treatment and combinations thereof.

  RNA processing: An intracellular mechanism that plays a role in RNA maturation, modification, and degradation; RNA splicing, intron excision, exosome digestion, nonsense-mediated decay (NMD) ), RNA editing, RNA processing and combinations thereof.

Target cell: a single or multiple human cells selected from the group consisting of somatic cells, tissues, stem cells, germline cells, teratomas cells, tumor cells, cancer cells and combinations thereof.
Cancer tissue: Neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor / brain cancer, lymphoma, leukemia and combinations thereof.

  Expression competent vectors: linear or circular single-stranded or double-stranded DNA selected from the group consisting of plasmids, viral vectors, transposons, retrotransposons, DNA transgenes, jumping genes and combinations thereof.

  Antibacterial genes for antibiotics: penicillin G, streptomycin, ampicillin (Amp), neomycin, G418, kanamycin, erythromycin, paromycin, fomycin (Phophomycin), spectromycin, tetracycline (Tet), doxycycline (Dox), rifampicin, amphotericin B, gentamycin, chloramphenicol ( A gene capable of degrading an antibiotic selected from the group consisting of chloramphenicol, cephalothin, tylosin and combinations thereof.

  Restriction / cloning site: DNA motif to restrict enzymatic degradation, AatII, AccI, AflII / III, AgeI, ApaI / LI, AseI, Asp718I, BamHI, BbeI, BclI / II, BglII, BsmI, Bsp120I, BspHI / LU11I / 120I, BsrI / BI / GI, BssHII / SI, BstBI / U1 / XI, ClaI, Csp6I, DpnI, DraI / II, EagI, Ecl136II, EcoRI / RII / 47III / RV, EheI, FspI, HaeIII, HhaI , HinPI, HindIII, HinfI, HpaI / II, KasI, KpnI, MaeII / III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvI Including, but not limited to, / II, RsaI, SacI / II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI.

  Gene delivery: from polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene gun penetration, and combinations thereof A genetic engineering method selected from the group consisting of

  Genetic engineering: DNA recombination method selected from the group consisting of DNA restriction enzyme reaction and conjugation reaction, homologous gene recombination, transgene integration, transposon insertion, jumping gene integration, retroviral infection, and combinations thereof is there.

  Cell cycle regulator: a cellular gene involved in the control of cell division and proliferation rate, CDK2, CDK4, CDK6, cyclin, BMI-1, p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1 and p27Kip1 and combinations thereof Including, but not limited to.

  Tumor suppressive effect: cell antitumor and / or anticancer mechanism and reaction, cell cycle attenuation, cell cycle arrest, tumor cell growth inhibition, cell tumorigenesis inhibition, tumor / cancer cell transformation inhibition Including, but not limited to, induction of apoptosis of tumor / cancer cells, induction of normal cell recovery, reprogramming of highly malignant cancer cells to a benign low-grade condition (tumor regression) and combinations thereof .

  Cancer therapeutic effect: Cell response and / or cell mechanism caused by drug treatment, suppression of oncogenic gene expression, suppression of cancer cell proliferation, suppression of cancer cell invasion and / or transition, suppression of cancer metastasis, cancer cell death Induction, prevention of tumor / carcinogenesis, prevention of cancer recurrence, suppression of cancer progression, cell repair of damaged tissue, reprogramming from advanced malignant cancer to more benign low-grade condition (cancer regression / mitigation) and its Including, but not limited to, combinations.

  Gene silencing effect: Cell response after suppression of gene function, suppression of oncogenic gene expression, suppression of cell proliferation, cell cycle arrest, tumor suppression, cancer regression, cancer prevention, cell apoptosis, This includes, but is not limited to, cell repair and / or recovery, cell reprogramming, reprogramming of diseased cells to a relatively normal state (spontaneous healing), and combinations thereof.

Cancer reversal: A reprogramming mechanism that restores advanced cancer malignant properties to a relatively normal, low-grade state in vitro, in vitro or in vivo.
Target cell: a single or multiple human cells selected from the group consisting of somatic cells, tissues, stem cells, germline cells, teratomas cells, tumor cells, cancer cells and combinations thereof.

Cancer tissue: Neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor / brain cancer, lymphoma, leukemia and combinations thereof.
Transcription inducer: A chemical agent that can induce and / or enhance eukaryotic RNA and / or gene transcription by a pol-2 or pol-2 family promoter in prokaryotic cells. For example, transcription inducers include MOPS, ethanol, glycerin and functional analogs thereof such as 2- (N-morpholino) ethanesulfonic acid (MES), 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid ( HEPES) and chemical structures similar to mannitol or mixtures thereof, including but not limited to.

Antibody: Peptides or protein molecules that encode a receptor having a preselected conserved domain structure and capable of binding to a preselected ligand.
Medicinal and / or therapeutic applications: diagnosis, stem cell generation, stem cell research and / or therapy development, tissue / organ repair and / or recovery, wound healing, tumor suppression, cancer treatment and / or prevention, Biomedical applications, devices and / or equipment used in disease treatment, drug production and combinations thereof.

B. Compositions and their applications Compositions to produce novel microRNA precursors (pro-miRNAs) produced by prokaryotes that can reprogram the malignant characteristics of human cancers to a low benign or normal-like state in vitro, in vitro and in vivo. And (a) at least one chemical inducer containing a structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin or mixtures thereof; b) a plurality of prokaryotic cells comprising at least one pre-miRNA coding gene via transcription driven by a eukaryotic pol-2 and / or pol-2 family promoter, and (a) and (b) , Mixed under conditions that induce the gene expression to produce pre-miRNA encoded in prokaryotic cells. It should be noted that chemical inducers can stimulate RNA transcription driven by eukaryotic promoters in prokaryotes.

  In principle, the present invention induces rapid adaptation of prokaryotes without the need to use a prone prokaryotic promoter with the wrong tendency or to incubate complicated and costly hybridoma or mammalian cells. Provide a novel composition design and its applicable strategies for directly expressing specific required microRNA precursors (pre-miRNA) using eukaryotic pol-2 and pol-2 family promoters.

  The prokaryote is a bacterial cell strain, in particular E. coli, and the chemical inducer may be 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin or a mixture thereof. preferable. The eukaryotic promoter may be a eukaryotic pol-2 promoter such as EF1α, or a pol-2 compatible (pol-2 class such as a cytomegalovirus (CMV) promoter or a retroviral long terminal repeat (LTR) promoter). It is also preferred that it is a viral promoter. The pre-miRNA coding gene via the eukaryotic promoter encodes non-coding and / or protein-encoding RNA transcripts (for example, gene transcripts containing introns). Non-coding and protein-encoding RNA transcripts or both (eg, gene transcripts containing introns) include microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA) and the like Selected from the group consisting of precursors and shRNA / siRNA homologues and combinations thereof. The protein-encoding RNA can be selected from, but not limited to, the group consisting of gene-encoding enzymes, growth factors, antibodies, insulin, botulinum toxins (botox), functional proteins and / or analogs thereof, and combinations thereof. The conditions for inducing the expression of the pre-miRNA coding gene are bacterial incubation conditions, for example, preferably a Luria-Bertani (LB) culture solution to which the chemical inducer is added at 37 ° C.

  The present invention or application document includes at least one color graph. After applying and paying the necessary costs, the USPTO will provide a copy of the invention or patent publication application with a color graph.

  For the purpose of illustrating the invention, reference is made to the drawings which are not intended to limit the invention. Explain as follows

FIG. 1A and FIG. 1B show an expression vector composition (1A) driven by a eukaryotic promoter and its expression mechanism (1B) used for the generation of RNA transcripts and / or proteins in prokaryotes. In order to verify the present invention, the new pLenti-EF1α-RGFP-miR302 vector (FIG. 1A) was transformed into E. coli DH5α competent cells with MOPS, glycerin and / or ethanol stimulation to produce RGFP protein and miR-302s. An example composition for producing the precursor (pre-miR-302s). pLenti-EF1α-RGFP-miR302 is a lentiviral plasmid vector designed by the present inventor and expressing various microRNAs / shRNAs, mRNAs and / or proteins / peptides in both prokaryotes and eukaryotes. As described in the present invention, by the mechanism (1B) described, one with ordinary knowledge in the field of technology to which the present invention belongs will use either microRNA / shRNA instead of miR-302 or RGFP It is easy to use any mRNA / protein instead of. Black arrows indicate pathways that appear in both prokaryotic and eukaryotic cells, and blank arrows indicate steps that appear only in eukaryotic cells. FIG. 2 shows the results of cultures incubating bacteria treated with a mixture of 0.1% (v / v) MOPS and 0.05% (v / v) glycerin (left) or untreated (right). E. coli competent cells were transformed with pLenti-EF1α-RGFP-miR302 prior to treatment with chemical inducers. FIG. 3 shows the results after different bacterial pellets were treated with 0.1% (v / v) MOPS. E. coli competent cells were transformed with pLVX-Grn-miR302 + 367 (green) or pLenti-EF1α-RGFP-miR302 (red) before treatment with MOPS. FIG. 4 shows the inducibility of various chemical inducers for the induction of gene expression driven by the pol-2 promoter in E. coli competent cells. In all of the test chemicals, three of the most potent inducers are MOPS, glycerin and ethanol. The concentration of chemical used may be in the range of about 0.001% to 4%, most preferably 0.01% to 1%. FIG. 5 shows the Western blot results of red RGFP protein expression induced by MOPS, glycerin and ethanol, respectively. Bacterial RuvB protein is used as a housekeeping standard to normalize detected RGFP expression. Protein extracted from blank E. coli cells (ie, not transformed with the vector) is used as a negative control. FIG. 6 shows the northern blot results of miR-302 family cluster (about 700 nt) and induced precursors (pre-miR-302s with 1 to 4 hairpins) induced by MOPS, glycerin and ethanol, respectively. Indicates. RNA extracted from blank E. coli cells is used as a negative control. FIG. 7 shows the generation of iPSCs using miR-302 and / or pre-miR-302 isolated from bacterial competent cell extracts (BE). It was demonstrated by Northern blot analysis as shown in FIG. As reported, miR-302 reprogrammed iPSCs (also referred to as mirPSCs) form a cluster of spherical cells and express strong Oct4 as a standard hESC mark. FIG. 8 shows the overall DNA desmethylation of the Oct4 and Sox2 gene promoters induced by miR-302 and / or pre-miR-302 isolated from bacterial competent cell extracts (BE). This was verified by Northern blot analysis as shown in FIG. As demonstrated by Simonsson and Gurdon (Nat Cell Biol. 6, 984-990, 2004), the two phenomena of global DNA desmethylation and Oct4 expression both reprogram somatic cells to form iPSCs. It is necessary to do. FIG. 9 shows an in vitro tumorigenicity analysis in which the human liver cancer cell line HepG2 responds to transfection with miR-302. Cells obtained after transfection with miR-302 are marked as mirPS-HepG2, indicating that their cancer cell properties change to an induced pluripotent stem cell (iPSC) -like state. Compare the changes in morphology and cell cycle rate before and after transfection with miR-302. The peak graph of flow cytometry analysis on cell morphology (n = 3, p <0.01) shows the DNA content of each cell relative to the cell cycle stage. Figures 10A and 10B show HPLC purification and analysis using synthetic standard uDNA (derived from Sigma-Genosys) and freshly extracted pro-miR-302s isolated from pLenti-EF1α-RGFP-miR302 transformed E. coli cells The results are shown. The standard uDNA is designed to correspond to natural pre-miR-302a, ie 5′-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3 ′ (SEQ.ID.NO.4). FIG. 11A and FIG. 11B show the results of microRNA (miRNA) microarray analysis using small RNA extracted from blank E. coli competent cells or pLenti-EF1α-RGFP-miR302 (RGFP-miR302) transfected cells. The extracted small RNA is further purified by HPLC as shown in the green mark area of FIG. 10B. FIG. 11A shows that RNA from blank E. coli cells exhibits very little microRNA (green spots mean not statistically significant, red spots indicate positive results). This is because prokaryotes do not have some essential enzymes required for microRNA expression and processing, such as Pol-2, Drosha and RNase III Dicer. Prokaryotic RNA polymerase does not efficiently transcribe small RNAs having a high secondary structure, such as hairpin pre-miRNAs and shRNAs. Therefore, as shown in FIG.11B, the inventor can express the expression of specific microRNAs in prokaryotic cells only by the present invention, for example, miR-302a, a *, b, b *, c, c *, d and d *. I can stimulate. Since prokaryotic cells do not have Dicer, all microRNAs will present their precursor structural form, for example, pri-miRNA (4 hairpin clusters) and / or pre-miRNA (1 hairpin precursor) Retained. Therefore, from the results of FIGS. 10B and 11B, (1) small RNA extracted from RGFP-miR302 transfected cells mainly contains pure miR-302 precursor and (2) E. coli competent cells Two facts have already been established that there is almost no other type of microRNA contamination. FIG. 12 shows expressed microRNA extracted from blank E. coli competent cells (first set, as shown in FIG. 11A) or pLenti-EF1α-RGFP-miR302 transfected cells (second set, as shown in FIG. 11B). A list of Signals below 500 are not statistically significant (shown in green in FIGS. 11A and 11B), which can be caused by low copy number expression or high background. Figures 13A and 13B show the sequencing results for the miR-302 family cluster (13A) and the individual pro-miR-302a, pro-miR-302b, pro-miR-302c and pro-miR-302d sequences (13B). Indicates. miR-302 family cluster (= pri-miR-302) results, 5'-AAUUUUUUUC UUCUAAAGUU AUGCCAUUUU GUUUUCUUUC UCCUCAGCUC UAAAUACUCU GAAGUCCAAA GAAGUUGUAU GUUGGGUGGG CUCCCUUCAA CUUUAACAUG GAAGUGCUUU CUGUGACUUU AAAAGUAAGU GCUUCCAUGU UUUAGUAGGA GUGAAUCCAA UUUACUUCUC CAAAAUAGAA CACGCUAACC UCAUUUGAAG GGAUCCCCUU UGCUUUAACA UGGGGGUACC UGCUGUGUGA AACAAAAGUA AGUGCUUCCA UGUUUCAGUG GAGGUGUCUC CAAGCCAGCA CACCUUUUGU UACAAAAUUU UUUUGUUAUU GUGUUUUAAG GUUACUAAGC UUGUUACAGG UUAAAGGAUU CUAACUUUUU CCAAGACUGG GCUCCCCACC ACUUAAACGU GGAUGUACUU GCUUUGAAAC UAAAGAAGUA AGUGCUUCCA UGUUUUGGUG AUGGUAAGUC UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA AAAUAACUUU AUUGUAUUGA CCGCAGCUCA UAUAUUUAAG CUUUAUUUUG UAUUUUUACA UCUGUUAAGG GGCCCCCUCU ACUUUAACAU GGAGGCACUU GCUGUGACAU GACAAAAAUA AGUGCUUCCA UGUUUGAGUG UGGUGGUUCC UACCUAAUCA GCAAUUGAGU UAACGCCCAC ACUGUGUGCA GUUCUUGGCU ACAGGCCAUU ACUGUUGCUA-3 '(SEQ.ID. NO.5), and the individual sequences of pro-miR-302a, pro-miR-302b, pro-miR-302c and pro-miR-302d are each 5′-CCA CCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3 '(SEQ.ID.NO.6), 5'-GCUCCCUUCA ACUUUAACUG GGAAGUGCUU UCUGUGAGACUUUACUGUGAGUGUCUUGG GUGAAACAAA AGUAAGUGCU UCCAUGUUUC AGUGGAGG-3 ′ (SEQ.ID.NO.8) and 5′-CCUCUACUUU AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU GAGUGUGG-3 ′ (SEQ.ID.NO.9). FIG. 14 shows the in vivo treatment results of a pre-IND test that uses human pro-miR-302 as an injecting drug to treat human liver cancer xenografts in SCID-beige nude mice. After 3 treatments (once a week), the pro-miR-302 drug (= pre-miR-302) reduced the cancer size from 728 ± 328 mm ³ (untreated blank, C) to 75 ± 15 mm Reduced well to ³ (processed by pro-miR-302, T). This indicates that the average size of the cancer has been reduced by about 90%. No significant therapeutic effect has been found in treatment with synthetic siRNA mimics (siRNA-302). Further histological examination (rightmost figure) shows that normal liver lobule-like structures (circles indicated by black arrows) are formed only in cancers treated with pro-miR-302, but not in other treatments or controls. This indicates that a reprogramming mechanism has occurred and the properties of malignant cancer cells can be restored to a relatively normal state (referred to as “cancer reversal”). FIG. 15 shows the histological similarity between normal liver tissue in vivo and human liver cancer xenografts treated with pro-miR-302. After 3 treatments (once a week), the pro-miR-302 drug successfully reprograms advanced (IV) human liver cancer transplants to a more benign (less than II) condition. Treated cancer implants are similar to normal liver tissue (top figure) and contain classic livers (CV) -like and portal trivial (PT) -like structures (indicated by black arrows) Can form lobule. Because cancer cells are usually more acidic than normal hepatocytes, the results of hematoxylin and eosin (H & E) staining showed a lot of purple in cancer cells, but a lot of red in normal hepatocytes showed that. FIG. 16 shows an untreated human liver cancer implant in SCID-beige nude mice, a human liver cancer implant treated with siRNA, a human liver cancer implant treated with pro-miR-302, and normal liver tissue. A histopathological comparison is shown. When untreated (top view), transplanted human liver cancer invasively invades normal tissues, such as muscle and blood vessels, and is a large cell-cell and cancer-tissue fusion structure. Which shows its malignant and highly metastatic nature. Treatment with siRNA mimic (siRNA-302) does not significantly reduce the malignancy of transplanted cancer (middle panel), which is likely due to the short half-life of siRNA. In contrast, treatment with pro-miR-302 not only reprogrammed the transplanted cancer to a relatively normal-like form (no fusion), but also significantly suppressed the cancer from entering the surrounding tissue ( Middle bottom diagram). Compared to normal liver tissue (bottom diagram), cancer treated with pro-miR-302 shows normal-like lobular structure, glandular cell organization and cell-cell and cancer-tissue binding sites. A clear boundary (black arrow) was formed, indicating that the malignancy of these treated cancers was reduced to a very benign state. FIGS. 17A and 17B show a comparison of healing results between wounds treated in vivo (17A) and treated with miR-302 (17B). The isolated miR-302 molecule (20-400 μg / mL) is combined with di- / tri-glycylglycerin, delivery reagent and antibiotic ointment to form the drug of choice. Used in the test for the local treatment of large open wounds of 2cm x 2cm on the skin of the pig's back in vivo (n = 6 in each group). After approximately 2 weeks of treatment (once a day), the wound that has been healed is dissected and further made into tissue sections and examined under a microscope. According to the data, the wound treated by miR-302 has no scar or very small scar (no scar) (top figure in Fig. 17B, n = 6/6), Almost all wounds that have not been treated (treated with antibiotic ointment alone) contain large scars (top view of FIG. 17A, n = 5/6). In addition, a large number of CD34 positive adult stem cell clusters (marked with green fluorescent antibody) were prominently observed in the wounds treated with miR-302 (the bottom figure in FIG. 17B, n = 6/6) However, it was not observed in untreated control wounds (bottom of FIG. 17A, n = 0/6). As can be seen from these results, pre-miR-302 induces proliferation and / or regeneration of CD34 positive adult stem cells to enhance tissue repair and regeneration, such as human degenerative diseases such as Alzheimer's disease, Parkinson It can have a very beneficial therapeutic effect on lesions due to disease, osteoporosis, diabetes and cancer. This therapeutic effect also contributes to reprogramming highly malignant cancers into low-grade benign and thus normal-like tissues, a novel mechanism called cancer reversal or cancer regression.

Illustration:
In the description of the following experiments, M (molar concentration); mM (molar concentration); μm (micromolar); mol (molar); pmol (picomolar); gm (gram); mg (milligram); ); Ng (nanogram); L (liter); ml (milliliter); μl (microliter); ° C (degree of Celsius); RNA (ribonucleic acid); DNA (deoxyribonucleic acid); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPES buffered saline); SDS (sodium dodecyl sulfate) Tris-HCl (tri-hydroxymethylaminomethane-hydrochloride); ATCC (American Type Culture Collection, Rockville, MD); hESC (human embryonic stem cells); and iPSC (induced pluripotent stem cells) ) Is applied.

1. Incubation and chemical treatment of bacterial cells
E. coli DH5α competent cells are obtained as part from a z-competent E. coli transformation kit (Zymo Research, Irvine, Calif.) and then pre-manufactured 5 μg of plasmid vector such as pLenti-EF1α-RGFP-miR302 or pLVX -Grn-miR302 + 367 was mixed and transformed. Untransformed cells were incubated normally in Luria-Bertani (LB) medium supplemented with 10 mM MgSO ₄ and 0.2 mM glucose under frequent agitation at 37 ° C. and 170 rpm. Meanwhile, the transformed cells were incubated in the above LB medium supplemented with an additional 100 μg / ml ampicillin. For chemical induction, 0.5 to 2 ml of MOPS, glycerin and / or ethanol, respectively or in combination, was added to a 1 L LB medium supplemented with 10 mM MgSO ₄ and 0.2 mM glucose and with 100 μg / ml ampicillin. For negative controls, transformed cells were incubated in LB broth supplemented with ampicillin as described above but without any chemical inducer. The results are shown in FIGS.

2. Incubation of human cells and transfection with MicroRNA The human hepatoma cell line HepG2 was obtained from ATCC and cultured according to the manufacturer's suggestions. For transfection, 15 μg pre-miR-302 was dissolved in 1 ml fresh RPMI medium and mixed with 50 μl X-tremeGENE HP DNA transfection reagent (Roche, Indianapolis, IN). After 10 minutes incubation, the mixture was added to a 100 mm cell dish containing 50-60% HepG2 cell confluency. The medium was renewed after 12-18 hours. After these transfected cells have formed a spherical iPSC cluster, the medium is made up of 20% gene knockout serum, 1% MEM non-essential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate. 10 ng / ml bFGF, 10 ng / ml FGF-4, 5 ng / ml LIF, 100 IU / ml penicillin / 100 μg / ml streptomycin, 0.1 μM A83-01 and 0.1 μM valproic acid (Stemgent, San Diego , CA) was replaced with gene knockout DMEM / F-12 medium (Invitrogen) supplemented with cells, and the cells were incubated at 37 ° C. in the presence of 5% CO ₂ . The results are shown in FIG.

3. Follow extraction and proposal Western blot analysis manufacturer of protein, protease inhibitors, leupeptin and (Leupeptin), TLCK, TAME and PMSF is supplemented with CelLytic-M decomposition / extraction reagent (Sigma) by the cell (10 ⁶⁾ Decomposed. The degradation product was centrifuged at 12,000 rpm for 20 minutes at 4 ° C., and the supernatant was recovered. Protein concentration was measured using a modified SOFTmax protein analysis set in an E-max microplate reader (Molecular Devices, CA). Add 30 μg of cell lysate to each SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, boil for 3 minutes, then 6-8% polyacrylamide Placed on gel. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted to nitrocellulose films, and incubated for 2 hours in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) at room temperature. The primary antibody was then added to the reagent and the mixture was incubated at 4 ° C. Primary antibodies include Oct3 / 4 (Santa Cruz Biotechnology, Santa Cruz, CA), RuvB (Santa Cruz) and RGFP (Clontech). After overnight, the film was rinsed 3 times with TBS-T and then to goat anti-mouse IgG conjugated secondary antibody (1: 2,000; Invitrogen-Molecular Probes) marked with Alexa Fluor 680 reactive dye at room temperature. Exposed for 1 hour. After three additional rinses with TBS-T, immunoblotting fluorescence scanning and image analysis were performed with Li-Cor Odyssey infrared imaging equipment and Odyssey software v.10 (Li-Cor). The results are shown in FIG.

4. RNA extraction and Northern blot analysis Total RNA (10 μg) was separated by mirVana ^™ miRNA isolation kit (Ambion, Austin, TX) and subjected to 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis And then electroblotted onto a nylon film. Detection of miR-302s and related pre-miR-302s was performed with [LNA] -DNA probe (5 ′-[TCACTGAAAC] ATGGAAGCAC TTA-3 ′) (SEQ. ID. NO. 10). The probe is purified by high performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of [ ³² P] -dATP (> 3000 Ci / mM, Amersham International, Arlington Heights, IL) It was done. The results are shown in FIG.

5. Plasmid amplification and plasmid DNA / total RNA extraction After transformation, E. coli DH5α competent cells (obtained from Example 1) were supplemented with 10 mM MgSO ₄ and 0.2 mM glucose in LB medium. Incubated at 37 ° C under frequent agitation at 170 rpm. To induce RNA transcription driven by a eukaryotic promoter, transformed cells were propagated overnight by adding 0.5-2 ml of MOPS, glycerin and / or ethanol per liter of LB culture. Amplified plasmid DNAs and expressed mRNAs in transformed cells according to the manufacturer's strategy, except that RNase A was not added to the P1 buffer using the HiSpeed Plasmid Purification Kit (Qiagen, Valencia, CA) / microRNAs were separated. The final extract containing both plasmids and mRNAs / microRNAs was then dissolved in ddH ₂ O treated with DEPC and stored at −80 ° C. before use. To purify only the amplified plasmid vector, RNase A was added to the P1 buffer and the extraction process was performed according to the manufacturer's strategy.

6. Separation / purification of MicroRNA and Pre-miRNA
To purify microRNAs and pre-miRNAs, mirVana ^™ miRNA isolation kit (Ambion, Austin, TX) was used to further extract total RNA isolated from Example 5 according to the manufacturer's strategy. The final product thus obtained was dissolved in ddH ₂ O treated with DEPC and stored at −80 ° C. before use. Bacterial RNAs naturally degrade very quickly (within a few hours), but most eukaryotic hairpin microRNA precursors (pre-miRNAs and pri-miRNAs) are stable at 4 ° C (half-life of 3-4) Inventors can take advantage of this half-life difference to obtain relatively pure pri- / pre-miRNAs for use in other applications. For example, as shown in FIG. 9, the pre-miR-302s thus obtained can be used to reprogram somatic cells into hESCs iPSCs.

7. Immunostaining analysis Tissue samples were embedded, sectioned and immunostained as previously reported (Lin et al., 2008). Primary antibodies include Oct4 (Santa Cruz) and RGFP (Clontech, Palo Alto, CA). Goat anti-rabbit or horse anti-mouse antibodies marked with a fluorescent dye were used as secondary antibodies (Invitrogen-Molecular Probes, Carlsbad, Calif.). Positive results were tested and analyzed at 100 × or 200 × magnification using the Metamorph imaging program (Nikon) on a fluorescent 80i microscopic quantification system. The result is shown in FIG.

8. DNA sequencing with sulfite
Genomic DNA is isolated from approximately 2,000,000 cells using a DNA isolation kit (Roche) and 1 μg of DNA isolated using sulfite (CpGenome DNA modification kit, Chemicon, Temecula, CA) according to the manufacturer's suggestion Were further processed. Sulphite treatment converted all unmethylated cytosine to uracil and retained methylated cytosine as cytosine. In DNA sequencing with sulfite, the inventors amplify the promoter region of the Oct4 gene with PCR primers, which are 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3 ′ (SEQ.ID.NO.11) and 5′-CCCTCCTGAC CCATCACCTC CACCACC-3 ′ (SEQ.ID.NO.12). In PCR, DNA modified with sulfite (50 ng) and primers (total 100 pmol) were mixed in 1 × PCR buffer, heated to 94 ° C., maintained for 2 minutes, and immediately cooled on ice. Thereafter, 25 PCR cycles were carried out using the Expand High Fidelity PCR kit (Roche) for 1 minute at 94 ° C. and 3 minutes at 70 ° C. The PCR product with the correct size was further fractionated by 3% agarose gel electrophoresis, purified by gel extraction filter (Qiagen) and then used for DNA sequencing. A detailed profile of the DNA methylation site was then obtained, as shown in FIG. 8, by comparing cytosine with no change in the converted DNA sequence with the unconverted DNA sequence.

9. DNA density flow cytometry cells were treated with trypsin to form aggregates and fixed for 1 hour by resuspension in 1 ml PBS containing 70% methanol pre-cooled at -20 ° C. Cells are formed as clumps, washed once with 1 ml PBS, and again as clumps, 1 ml containing 1 mg / ml propidium iodide, 0.5 μg / ml RNase at 37 ° C Resuspended in PBS for 30 minutes. Thereafter, about 15,000 cells were analyzed in BD FACSCalibur (San Jose, CA). Cell doublets were eliminated by plotting a graph of pulse width versus pulse area and gating single cells. The software package Flowjo was used to analyze the data collected by the “Watson Pragmatic” algorithm. The result is shown in the top diagram of FIG.

10. Microarray analysis of microRNA (miRNA) Small RNA derived from each cell culture was separated using a mirVana ^™ miRNA isolation kit (Ambion) at a cell density of about 70%. The purity and amount of separated small RNAs were measured and evaluated by 1% formaldehyde-agarose gel electrophoresis and spectrophotometer (Bio-Rad), and immediately frozen in dry ice and LC Sciences (San Diego, CA) and used for miRNA microarray analysis. Each microarray chip was hybridized and a single sample was marked with Cy3 or Cy5, or a pair of samples were marked with Cy3 and Cy5, respectively. Background removal and standardization were performed according to the manufacturer's suggestions. In the analysis of the two samples, a p-value calculation was performed to generate a list of transcripts (yellow-red signal) expressed with a difference greater than 3 fold. The final microarray results are shown in FIGS. 11A and 11B, the list of differentially expressed microRNAs is shown in FIG. 12, and small RNAs extracted from blank E. coli cell lysates (Group 1) and pLenti-EF1α-RGFP -Comparison with small RNA (group 2) extracted from miR302 transformed cell lysate.

11. In vivo liver cancer therapy test Xenotransplantation of human liver cancer into immunocompromised SCID-beige mice is an effective animal model for studying liver cancer metastasis and therapy. To construct this model, the inventor mixed 5 million human liver cancer (HepG2) cells with 100 μL of matrix gel and transplanted the mixture subcutaneously on each flank of the hind limb of the mouse. Therefore, approximately the same amount of cancer cells was transplanted on both sides of the hind limb of the mouse. About 2 weeks after transplantation, cancer was observed, and its average size was about 15.6 ± 8 mm ³ (initial size of cancer before treatment). For each mouse, the inventor selected one side with larger size cancer as the treatment group and the other side with smaller cancer as the control group. Since the same mouse was treated with a blank formulation reagent (negative control) on one side and a compounded drug (pro-miR-302) on the other side, the results obtained in this way could be due to individual differences Changes can be minimized.

  In order to deliver pro-miR-302 to the target cancer region in vivo, the inventors ordered a professional formulation company Latitude (San Diego, Calif.), And pro-miR-302s with liposomes having a diameter of 160-200 nm Encapsulated in nanoparticles. Nanoparticles containing these pro-miR-302 have been tested to be almost 100% stable at room temperature for more than 2 weeks and almost 100% for more than 1 month at 4 ° C. All of the other synthetic siRNA mimics (siRNA-302) rapidly superdegrade in 50% within 3-5 days under the same conditions, which means that pro-miRNA, not siRNA, is sufficiently stable and therapeutic Can be used as In the toxicity analysis, the inventors further injected 300 μL of formulated pro-miR-302 (1 mg / mL) maximally into the tail vein (n = 8) of each mouse. After 6 months, none of the mice tested had any detectable side effects. In general, unmodified ribonucleic acids are relatively non-immunogenic and are easily metabolized by tissue cells, providing a safe tool for in vivo therapy.

To test the effect of the drug, the inventor injected 200 μL of formulated pro-miR-302 subcutaneously on one side of the mouse and 200 μL of blank compounded reagent on the other side, and continued three times in the same injection mode (Inject once a week). Drugs and reagents were applied to the surrounding area of the cancer site and were absorbed into the cancer and its surrounding tissue within 18 hours. Samples were collected one week after the third injection. Heart, liver, kidney and transplanted cancer were removed for use in further histology. Tumor formation was monitored by palpation and tumor volume was calculated by the equation (length x width ² ) / 2. Tumor nests were counted, dissected and weighed, and histological examination was performed on them by H & E and immunostaining analysis. Histological examination showed no detectable tissue nest in the heart, liver and kidney. The results are shown in FIG. 14, FIG. 15 and FIG.

12. Statistical analysis Any change in signal intensity greater than 75% in immunostaining, Western blot and Northern blot analysis was considered a positive result and was analyzed and presented as mean ± SE . Statistical analysis of the data was performed by one-way ANOVA. When the main effect was significant, Dunnett's post-hoc test identified groups that were significantly different from controls. A two-tailed student t test was used to make a pairwise comparison between the two treatment groups. For experiments involving more than one treatment group, ANOVA was performed followed by a post-hoc multiple range test. A probability is considered significant if p <0.05. All p-values were measured by a two-sided test.

Prior art documents
1. Lin SL and Ying SY. (2006) Gene silencing in vitro and in vivo using intronic microRNAs. Ying SY. (Ed.) MicroRNA protocols. Humana press, Totowa, New Jersey, pp. 295-312.
2. Lin SL, Chang D and Ying SY. (2006) Transgene-like animal models using intronic microRNAs. Ying SY. (Ed.) MicroRNA protocols. Humana press, Totowa, New Jersey, pages 321-334.
3. Lin SL, Chang D, Chang-Lin S, Lin CH, Wu DTS, Chen DT and Ying SY. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA 14, 2115 -2124.
4. Lin SL and Ying SY. (2008) Role of mir-302 microRNA family in stem cell pluripotency and renewal. Ying SY. (Ed.) Current Perspectives in MicroRNAs. Springer Publishers press, New York, pp. 167-185.
5. Lin SL, Chang D, Ying SY, Leu D and Wu DTS. (2010) MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 / 6 cell cycle pathways.Cancer Res. 70, 9473-9482.
6. Lin SL, Chang D, Lin CH, Ying SY, Leu D and Wu DTS. (2011) Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 39, 1054-1065.
7. Simonsson S and Gurdon J. (2004) DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat Cell Biol. 6, 984-990.
8. Takahashi et al. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663--676.
9. Wang et al. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478--1483.
10. Wernig et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318--324.
11. Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917--1920.
12. Buechler US Pat. No. 7,959,926.
13. US Patent No. 7,968,311 to Mehta.
14. Lin's European Patent No. EP 2198025.
15. Lin's US Patent Application No. 12 / 149,725.
16. Lin's US Patent Application No. 12 / 318,806.
17. Lin's US Patent Application No. 12 / 792,413.
18. Lin's US Patent Application No. 13 / 572,263.
19. Lin's US Patent Application No. 13 / 964,705.

Sequence listing
(1) General information:
(iii) Number of sequences: 12
(2) Information on SEQ ID NO: 1:
(i) Sequence features:
(A) Length: 23 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: RNA
(A) Explanation: / desc = "Composition"
(iii) Virtual: NO
(iv) Antisense: YES
(xi) Sequence description: SEQ ID NO: 1:
UCACCAAAAC AUGGAAGCAC UUA 23
(3) Information on SEQ ID NO: 2:
(i) Sequence features:
(A) Length: 23 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: RNA
(A) Explanation: / desc = "Natural"
(iii) Virtual: NO
(iv) Antisense: YES
(xi) Sequence Description: SEQ ID NO: 2:
ACUUAAACGU GGAUGUACUU GCU 23
(4) Information on SEQ ID NO: 3:
(i) Sequence features:
(A) Length: 17 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: RNA
(A) Explanation: / desc = "Natural"
(iii) Virtual: NO
(iv) Antisense: NO
(xi) Sequence Description: SEQ ID NO: 3:
UAAGUGCUUC CAUGUUU 17
(5) Information on SEQ ID NO: 4:
(i) Sequence features:
(A) Length: 69 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Hairpin
(ii) Molecular type: other nucleic acids
(A) Explanation: / desc = "Composition"
(iii) Virtual: YES
(iv) Antisense: NO
(xi) Sequence Description: SEQ ID NO: 4:
CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC
UUCCAUGUUU UGGUGAUGG 69
(6) Information on SEQ ID NO: 5:
(i) Sequence features:
(A) Length: 720 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Multiple hairpins
(ii) Molecular type: RNA
(A) Explanation: / desc = "Recombination"
(iii) Virtual: NO
(iv) Antisense: NO
(xi) Sequence description: SEQ ID NO: 5:
AAUUUUUUUC UUCUAAAGUU AUGCCAUUUU GUUUUCUUUC UCCUCAGCUC
UAAAUACUCU GAAGUCCAAA GAAGUUGUAU GUUGGGUGGG CUCCCUUCAA
CUUUAACAUG GAAGUGCUUU CUGUGACUUU AAAAGUAAGU GCUUCCAUGU
UUUAGUAGGA GUGAAUCCAA UUUACUUCUC CAAAAUAGAA CACGCUAACC
UCAUUUGAAG GGAUCCCCUU UGCUUUAACA UGGGGGUACC UGCUGUGUGA
AACAAAAGUA AGUGCUUCCA UGUUUCAGUG GAGGUGUCUC CAAGCCAGCA
CACCUUUUGU UACAAAAUUU UUUUGUUAUU GUGUUUUAAG GUUACUAAGC
UUGUUACAGG UUAAAGGAUU CUAACUUUUU CCAAGACUGG GCUCCCCACC
ACUUAAACGU GGAUGUACUU GCUUUGAAAC UAAAGAAGUA AGUGCUUCCA
UGUUUUGGUG AUGGUAAGUC UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA
AAAUAACUUU AUUGUAUUGA CCGCAGCUCA UAUAUUUAAG CUUUAUUUUG
UAUUUUUACA UCUGUUAAGG GGCCCCCUCU ACUUUAACAU GGAGGCACUU
GCUGUGACAU GACAAAAAUA AGUGCUUCCA UGUUUGAGUG UGGUGGUUCC
UACCUAAUCA GCAAUUGAGU UAACGCCCAC ACUGUGUGCA GUUCUUGGCU
ACAGGCCAUU ACUGUUGCUA 720
(7) Information on SEQ ID NO: 6:
(i) Sequence features:
(A) Length: 69 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Hairpin
(ii) Molecular type: RNA
(A) Explanation: / desc = "Recombination"
(iii) Virtual: YES
(iv) Antisense: NO
(xi) Sequence description: SEQ ID NO: 6:
CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC
UUCCAUGUUU UGGUGAUGG 69
(8) Information on SEQ ID NO: 7:
(i) Sequence features:
(A) Length: 73 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Hairpin
(ii) Molecular type: RNA
(A) Explanation: / desc = "Recombination"
(iii) Virtual: YES
(iv) Antisense: NO
(xi) Sequence Description: SEQ ID NO: 7:
GCUCCCUUCA ACUUUAACAU GGAAGUGCUU UCUGUGACUU UAAAAGUAAG
UGCUUCCAUG UUUUAGUAGG AGU 73
(9) Information on SEQ ID NO: 8:
(i) Sequence features:
(A) Length: 68 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Hairpin
(ii) Molecular type: RNA
(A) Explanation: / desc = "Recombination"
(iii) Virtual: YES
(iv) Antisense: NO
(xi) Sequence Description: SEQ ID NO: 8:
CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA AGUAAGUGCU
UCCAUGUUUC AGUGGAGG 68
(10) Information on SEQ ID NO: 9:
(i) Sequence features:
(A) Length: 68 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Hairpin
(ii) Molecular type: RNA
(A) Explanation: / desc = "Recombination"
(iii) Virtual: YES
(iv) Antisense: NO
(xi) Sequence description: SEQ ID NO: 9:
CCUCUACUUU AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC
UUCCAUGUUU GAGUGUGG 68
(11) Information on SEQ ID NO: 10:
(i) Sequence features:
(A) Length: 23 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: LNA-DNA hybrid
(A) Explanation: / desc = "Composition"
(iii) Virtual: YES
(iv) Antisense: YES
(xi) Sequence Description: SEQ ID NO: 10:
TCACTGAAAC ATGGAAGCAC TTA 23
(12) Information on SEQ ID NO: 11:
(i) Sequence features:
(A) Length: 17 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: other nucleic acids
(A) Explanation: / desc = "Composition"
(iii) Virtual: YES
(iv) Antisense: YES
(xi) Sequence Description: SEQ ID NO: 11:
GAGGCTGGAG CAGAAGGATT GCTTTGG 27
(13) Information on SEQ ID NO: 12:
(i) Sequence features:
(A) Length: 17 base pairs
(B) Type: Nucleic acid
(C) Chain type: single chain
(D) Topology: Linear
(ii) Molecular type: other nucleic acids
(A) Explanation: / desc = "Composition"
(iii) Virtual: YES
(iv) Antisense: YES
(xi) Sequence description: SEQ ID NO: 12:
CCCTCCTGAC CCATCACCTC CACCACC 27

Claims (15)

(A) providing at least one microRNA precursor comprising SEQ.ID.NO.3;
(B) providing a cell matrix comprising at least one CD34 positive cell;
(C) contacting the microRNA precursor of (a) with the cell matrix of (b), inducing proliferation or regeneration of the CD34 positive cells,
A method for inducing proliferation or regeneration of CD34-positive adult stem cells using a microRNA precursor (pre-miRNA).   2. The method of claim 1, wherein the microRNA precursor is generated by driving RNA transcription by a eukaryotic promoter in prokaryotic cells.   Driving RNA transcription by the eukaryotic promoter involves chemical agents containing the molecular structure of 3-morpholinopropane-1-sulfonic acid (MOPS), and SEQ.ID.NO.8, SEQ.ID.NO.7. Or at least one transformed prokaryotic cell carrying at least one expression vector encoding the sequence of SEQ.ID.NO.8, or SEQ.ID.NO.9. The method described in 1.   4. The method of claim 3, wherein the expression vector is a recombinant plasmid encoding the sequence of SEQ.ID.NO.5.   4. The method according to claim 3, wherein the expression vector is pLenti-EF1alpha-RGFP-miR302 containing a cytomegalovirus (CMV) or mammalian EF1α (EF1alpha) promoter, or both simultaneously.   3. The method according to claim 2, wherein the prokaryotic cell is an E. coli competent cell.   The microRNA precursor comprises at least one hairpin-type sequence of SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, or SEQ.ID.NO.9. The method according to 1.   2. The method according to claim 1, wherein the microRNA precursor is a kind of miR-302 precursor (pre-miR-302) produced by prokaryotes.   9. The method of claim 8, wherein the pre-miR-302 comprises at least one sequence of miR-302a, miR-302b, miR-302c, or miR-302d.   9. The method of claim 8, wherein the pre-miR-302 can be used as part of a drug component for pharmaceutical and therapeutic applications.   2. The method of claim 1, wherein the microRNA precursor can be used as part of a drug component for pharmaceutical and therapeutic applications.   2. The method of claim 1, wherein the proliferation of CD34 positive adult stem cells is useful for pharmaceutical and therapeutic applications.   13. The method of claim 12, wherein the proliferation of CD34 positive adult stem cells helps reprogram the malignant properties of human cancer cells in the body to a low grade benign state or a state similar to normal.   13. The method of claim 12, wherein the proliferation of the CD34 positive adult stem cells helps promote healing of a scar free wound in the body. 2. The method of claim 1, wherein the CD34 positive cells include skin, hair, muscle, blood (hematopoietic cells), stromal cells, and neural stem cells, or a combination thereof.