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CN117757789A - siRNA for inhibiting tumor growth and application thereof - Google Patents

siRNA for inhibiting tumor growth and application thereof Download PDF

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CN117757789A
CN117757789A CN202211176786.3A CN202211176786A CN117757789A CN 117757789 A CN117757789 A CN 117757789A CN 202211176786 A CN202211176786 A CN 202211176786A CN 117757789 A CN117757789 A CN 117757789A
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许强
王冠
魏金旺
王堃
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Genomicare Biotechnology Shanghai Co ltd
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Abstract

The invention relates to siRNA for inhibiting tumor growth and application thereof. Wherein the siRNA targeted to a gene comprising the sequence represented by SEQ ID NO:45 or SEQ ID NO:46 and methods of using such sirnas to inhibit tumor growth.

Description

siRNA for inhibiting tumor growth and application thereof
Technical Field
The invention relates to a method for inhibiting tumor cell growth by using allele specific siRNA targeting AURKB, siRNA for inhibiting tumor growth and application thereof.
Background
Tumor is one of the main causes of death worldwide, and as a large population in the world, chinese tumor data is not optimistic, and new cases and death cases growing gradually promote China to need more tumor prevention and treatment intervention to reduce the burden of future tumors.
Single Nucleotide Polymorphism (SNP) refers to a polymorphism in a DNA sequence that occurs due to variation in a single nucleotide position on a chromosomal sequence, and occurs more than 1% frequently in the population. Both genotypes comprising SNPs are alleles. Tumor cells frequently undergo loss of heterozygosity (LOH), which means that the LOH-producing tumor cells can only rely on one of the genotypes. If the gene in which LOH occurs is also an essential gene, we can specifically inhibit the expression of this genotype, thus killing tumor cells, while normal cells can still rely on another genotype to normally express the essential gene without being affected because LOH does not occur.
AURKB is a mitotic serine/threonine protein kinase belonging to the aurora kinase family. Aurora kinases were discovered since 1995 and were not first applied to the observation of human cancerous tissue expression until 1998, and are involved in spindle formation, centrosome maturation, chromosome differentiation, and cytokinesis during mitosis. AURKB is a member of the chromosomal passenger protein complex and plays an important role in the progression of the cell cycle. Deregulation of AURKB is observed in some tumors, and this gene is highly expressed in a variety of tumors and is associated with invasion, metastasis and resistance of tumor cells. Aurora kinase B (AURKB) has become an attractive drug target as an important tumor target, leading to the development of related small molecule inhibitors. Although AURKB drug development has been for many years, up to 30 AURKB inhibitors are currently in preclinical or clinical studies, but no drug has yet been marketed.
Small interfering RNAs (sirnas) are a class of 19-30 nucleotide long double-stranded RNA molecules that specifically degrade messenger ribonucleic acid (mRNA) by binding to an RNA-induced silencing complex (RISC), thereby reducing gene expression levels. In recent years, siRNA has become a popular field for drug development due to the characteristics of good specificity, high efficiency, easy development, repeated use and the like, and a plurality of siRNA drugs are currently marketed in batches.
Currently, many small molecule inhibitors against the aurrb target are in preclinical and clinical studies, but on the one hand, the drugs are not sufficiently specific and inhibit other kinase activities, and on the other hand, the side effects of the drug administration are significant. Thus, new inhibitors or inhibition methods need to be developed.
Disclosure of Invention
In view of this, the present invention provides an siRNA that specifically targets the expression of AURKB genes of a specific genotype, thereby achieving the goal of eliminating tumor cells without affecting normal cell function. The siRNA designed according to the SNP can specifically inhibit and kill tumor cells with LOH, while the growth of heterozygous cells (representing normal cells) is not obviously affected. Therefore, the region can become a novel siRNA drug targeting region for inhibiting tumor growth, thereby achieving the aims of good specificity, high efficiency, easy development, repeated use and the like of the siRNA drug.
The invention aims to solve the problems of insufficient specificity and obvious side effects of the small molecule inhibitor in the current stage. By designing two haplotype sequences of different siRNA targeted AURKB genes respectively, tumor cells expressing the haplotype can be specifically inhibited, so that the influence on heterozygous normal cells is reduced. Thus, the problem of high toxicity caused by the fact that the small molecule AURKB inhibitor cannot distinguish genotypes can be solved. And the two SNPs in the two haplotypes H1 (CGTGCCCAT) (SEQ ID NO: 45) and H2 (AGTGCCCAG) (SEQ ID NO: 46) were separated by only 7bp. Therefore, the region is very suitable as an siRNA targeting region, and can improve the specificity of siRNA, thereby inhibiting the growth of tumor cells with heterozygosity loss.
The terms "small interfering nucleic acid", "siNA" or siNA "molecule," small interfering RNA "," siRNA "," small interfering nucleic acid molecule "," small interfering oligonucleotide molecule "as used herein refer to any nucleic acid molecule capable of inhibiting or down-regulating gene expression by RNA interference mechanisms.
The term "target sequence" as used herein refers to a contiguous portion of the nucleotide sequence of an mRNA molecule (including messenger RNA (mRNA), which is the product of an RNA processing primary transcript product) formed during transcription of the H1/H2 gene. The target portion of the sequence will be at least long enough to act as a substrate for siRNA directed cleavage at or near this portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all subranges therebetween. As one non-limiting example, the target sequence can have a length of from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-21, or 21-22 nucleotides.
The term "RNA" as used herein refers to a molecule comprising at least one ribonucleotide residue, which includes double stranded RNA, single stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, and altered RNA or naturally occurring analogues of RNA.
The term "siRNA" or "dsRNA" as used herein refers to an iRNA comprising an RNA molecule or molecular complex having a hybridization duplex region comprising two anti-parallel and substantially complementary nucleic acid strands that are referred to as having "sense" and "antisense" orientations with respect to a target RNA. The duplex region may have any length that allows for specific degradation of the desired target RNA via the RISC pathway, but will generally range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. In the case of a duplex between 9 and 36 base pairs, the duplex may have any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any subrange therebetween, including, but not limited to, 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. Dsrnas produced in cells by Dicer and similar enzymatic processes typically have base pair lengths ranging from 19 to 22. One strand of the duplex region of dsDNA comprises a sequence that is substantially complementary to a region of the target RNA. The two strands forming the duplex structure may be from a single RNA molecule having at least one self-complementary region, or may be formed from two or more separate RNA molecules. Where the duplex region consists of two strands of a single molecule, the molecule may have duplex regions separated by a single nucleotide strand (referred to herein as a "hairpin loop") between the 3 '-end of one strand and the 5' -end of the respective other strand forming the duplex structure. The hairpin loop may comprise at least one unpaired nucleotide; in some embodiments, the hairpin loop may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 31, or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are made up of separate RNA molecules, these molecules need not be, but may be, covalently linked. In the case where the two chains are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker". The term "siRNA" is also used herein to refer to dsRNA as described above.
The term "inhibit" as used herein means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more protein subunits, is up-regulated or down-regulated such that the expression, level or activity is greater or less than that observed in the absence of a modulator. For example, in certain instances, AURKB gene expression is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an siRNA characterized in the present invention. In some embodiments, the AURKB gene is inhibited by at least about 60%, 70% or 80% by administration of an siRNA as described herein. In some embodiments, the AURKB gene is inhibited by administration of an siRNA as described herein by at least about 85%, 90%, 95%, 98%, 99%, or more.
The term "gene" as used herein refers to a nucleic acid encoding an RNA sequence, which includes, but is not limited to, structural genes encoding polypeptides.
Unless specifically indicated otherwise, "a" means "one (species)" or "a plurality (species)".
While some embodiments of the invention focus on siRNA, the disclosure herein is not to be construed as limited to siRNA, but also encompasses related compositions and methods implemented with small nucleic acid molecules, double stranded RNA (dsRNA), small RNA (mRNA), deoxyribonucleic acid interference (DNAi), and small hairpin RNA (shRNA) molecules, enzymatic nucleic acid molecules (Enzymatic Nucleic Acid), or antisense nucleic acid molecules.
In a first aspect, the invention relates to an siRNA targeted by a nucleic acid comprising SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
According to a preferred embodiment, wherein at least one strand consists of a nucleotide chain of 19-30 nucleotides in length, preferably of 19-29, 19-28, 19-27, 19-26, 19-25 nucleotides in length.
According to a preferred embodiment, wherein said siRNA comprises at least one modified nucleotide.
In one embodiment, wherein at least one of the modified nucleotides is selected from the group consisting of: 2 '-O-methyl modified nucleotides, nucleotides comprising a 5' -phosphorothioate group, terminal nucleotides linked to cholestene derivatives or dodecanoic acid didecarboxamide groups.
In one embodiment, wherein at least one of the modified nucleotides is selected from the group consisting of: 2' -deoxy modified nucleotides, locked nucleotides, non-base nucleotides, 2' -amino modified nucleotides, 2' -alkyl modified nucleotides, morpholino nucleotides, phosphoramidate nucleotides and nucleotides comprising non-natural bases.
According to a preferred embodiment, wherein at least one strand of said siRNA comprises a 3' overhang of at least one nucleotide.
According to a preferred embodiment, wherein the 3' end of each strand of said siRNA is modified with a pendant base.
According to a preferred embodiment, wherein the overhanging base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
In one embodiment, wherein the overhang base consists of any two deoxyribonucleotides in dA, dT, dG, dC.
According to a preferred embodiment, wherein said siRNA has a structure selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, a step of; SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; SEQ ID NO:7 and SEQ ID NO:8, 8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20, a step of; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process; SEQ ID NO:39 and SEQ ID NO:40, a step of performing a; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
in a second aspect, the invention relates to a vector comprising an siRNA according to the first aspect of the invention.
In a third aspect, the invention relates to a cell comprising an siRNA according to the first aspect of the invention.
In a fourth aspect, the invention relates to a pharmaceutical composition for inhibiting tumor growth comprising an siRNA according to the first aspect of the invention and a pharmaceutically acceptable excipient.
According to a preferred embodiment, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
In a fifth aspect, the invention relates to a pharmaceutical composition for inhibiting tumor growth comprising an siRNA that inhibits expression of AURKB and a pharmaceutically acceptable excipient.
According to a preferred embodiment, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
In a sixth aspect, the invention relates to a method for reducing AURKB expression in a target cell by administering an siRNA according to the first aspect of the invention.
In a seventh aspect, the present invention relates to a method of inhibiting tumor growth by administering to a subject in need thereof an siRNA, wherein the siRNA is targeted by a nucleic acid comprising the sequence of SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
According to a preferred embodiment, wherein at least one strand of said siRNA consists of a 19-30 nucleotide chain, preferably a 19-29, 19-28, 19-27, 19-26, 19-25 nucleotide chain in length.
According to a preferred embodiment, wherein said siRNA comprises at least one modified nucleotide.
According to a preferred embodiment, wherein the 3' end of each strand of said siRNA is modified with a pendant base.
According to a preferred embodiment, wherein the overhanging base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
In one embodiment, wherein the overhang base consists of any two deoxyribonucleotides in dA, dT, dG, dC.
According to a preferred embodiment, wherein said siRNA has a structure selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, a step of; SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; SEQ ID NO:7 and SEQ ID NO:8, 8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20, a step of; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process; SEQ ID NO:39 and SEQ ID NO:40, a step of performing a; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
according to a preferred embodiment, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
In an eighth aspect, the invention relates to a method of inhibiting tumor growth comprising administering to a subject in need thereof an siRNA that inhibits AURKB expression.
According to a preferred embodiment, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
In a ninth aspect, the invention relates to the use of an siRNA for the preparation of a medicament for inhibiting tumor growth, said siRNA targeting a polypeptide comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
According to a preferred embodiment, wherein at least one strand of said siRNA consists of a 19-30 nucleotide chain, preferably a 19-29, 19-28, 19-27, 19-26, 19-25 nucleotide chain in length.
According to a preferred embodiment, wherein said siRNA comprises at least one modified nucleotide.
According to a preferred embodiment, wherein the 3' end of each strand of said siRNA is modified with a pendant base.
According to a preferred embodiment, wherein the overhanging base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
In one embodiment, wherein the overhang base consists of any two deoxyribonucleotides in dA, dT, dG, dC. According to a preferred embodiment, wherein said siRNA has a structure selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4, a step of; SEQ ID NO:5 and SEQ ID NO:6, preparing a base material; SEQ ID NO:7 and SEQ ID NO:8, 8; SEQ ID NO:9 and SEQ ID NO:10; SEQ ID NO:11 and SEQ ID NO:12; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:18; SEQ ID NO:19 and SEQ ID NO:20, a step of; SEQ ID NO:21 and SEQ ID NO:22; SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:27 and SEQ ID NO:28; SEQ ID NO:29 and SEQ ID NO:30; SEQ ID NO:31 and SEQ ID NO:32; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:35 and SEQ ID NO:36; SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process; SEQ ID NO:39 and SEQ ID NO:40, a step of performing a; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
according to a preferred embodiment, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
In one embodiment, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
The excellent technical effects of the siRNA, the composition and the application thereof are mainly as follows:
(1) The specificity is high: by designing two haplotype sequences of different siRNA targeted AURKB genes respectively, tumor cells expressing the haplotype can be specifically inhibited, and two SNPs in the two haplotypes H1 and H2 are separated by only 7bp. Thus, targeting this region can increase the specificity of siRNA, thereby alleviating the effects on heterozygous normal cells.
(2) Easy to develop and reusable: the aim of eliminating tumor cells without affecting normal cell functions is achieved by specifically targeting the expression of AURKB genes with specific genotypes. The region can become a novel siRNA drug targeting region for inhibiting tumor growth, thereby achieving the aims of good specificity, high efficiency, easy development, repeated use and the like of the siRNA drug.
(3) Side effects and toxicity were weak: the small molecule AURKB inhibitor can not distinguish genotype and has large toxicity, and the siRNA of the invention can distinguish two haplotypes H1 and H2 so as to well solve the problems.
Brief description of the drawings
Fig. 1: AURKB is an essential gene in a variety of tumors. CAS-9 values less than 0 indicate inhibition of tumor growth, less than-3 indicate rapid death of tumor cells after the gene knockout, the gene being an essential gene;
fig. 2: compared with normal tissue expression, AURKB in liver cancer, lung cancer and glioma has obvious up-regulation of expression quantity;
fig. 3: the overall survival prognosis for patients with high AURKB expression in multiple cancer species is poor. Wherein FIG. 3A shows star data; FIG. 3B shows the TCGA data;
fig. 4: the newly discovered siRNA targetable region in AURKB (nm_004217) gene is located between chromosome 17 8205000-8205040 (grch 38.p14, nc_ 000017.11). The box positions are two SNP loci, included in the region;
fig. 5A-5C: siRNA transfection results;
fig. 6: cell Titer-Glo (CTG) method Cell viability assay results.
Detailed Description
Example 1:AURKB is an essential gene for a variety of tumors
AURKB was found to belong to an essential gene (contained in the Achilles_common_essentials essential gene list) based on tumor cell CRISP-Cas9 data. In addition, it was confirmed from the DepMap database CRISPR knockout test data that after AURKB is knocked out in brain cancer, lung cancer and liver cancer cell lines, the CRISPR scores were all negative and substantially less than-2 (FIG. 1), and thus AURKB gene was an essential gene in various tumors such as hepatocellular carcinoma, lung cancer, brain cancer, etc.
Example 2:in liver cancer, lung cancer and brain cancer, AURKB is up-regulated in tumor tissue expression level
TCGA data showed that AURKB expression levels were higher in both liver and lung cancer than in normal tissues. AURKB genes are selected from published website GEPIA2 (http:// gepia2.Cancer-pku. Cn/# analysis), corresponding cancer seeds are selected, and after Box Plot is selected, the web page can display the expression comparison diagram of AURKB genes of tumor and normal tissues. Compared with normal tissue expression, AURKB in liver cancer, lung cancer and glioma has significant up-regulation of expression quantity. Shows that AURKB has an affinity for tumorigenesis (fig. 2).
Example 3:GC and TCGA data show that the patients with hepatocellular carcinoma with low AURKB expression and lung cancer and brain cancer have better overall survival
The collar-star data showed that patients with low AURKB gene expression survived better overall than those with high AURKB gene expression in chinese brain cancer and liver cancer patients (fig. 3A). Meanwhile, TCGA data (http:// gepia2.Cancer-pku. Cn/# survivin) showed the same trend in patients with lung, liver and brain cancers, and overall survival was superior to those with low AURKB gene expression (grouped in median) compared to those with high AURKB expression (FIG. 3B). The results suggest that knockdown AURKB may extend the overall survival of the patient.
Example 4:novel siRNA targetable region of AURKB gene
SNP haplotypes were found on the AURKB exons, and the newly found siRNA targetable region in the AURKB (NM_004217) gene was located between chromosome 17 8205000-8205040 (GRCh38.p14, NC_ 000017.11), which is a completely novel siRNA targeting region (FIG. 4), and as a result, it was shown that two SNPs 7bp apart significantly improved the inhibition efficiency of siRNA.
In addition, the gene data of the collar star chinese population show that the proportion of chinese cancer population with heterozygous deletion of AURKB is greater than 20%, as shown in table 1 below.
TABLE 1 Haplotype LOH crowd ratio
Hepatocellular carcinoma Lung cancer Brain cancer
27.8％ 23.4％ 21.2％
Example 5:design and inhibition efficiency of siRNA
According to the target region and SNP information, a series of siRNAs which can cover two genotypes are designed and subjected to transfection test, and the interference efficiency is observed.
A series of the above sirnas designed for this region were screened to obtain sirnas with good knockdown efficiency, which were series of siRNA sequences designed for haplotype H1 (CGTGCCCAT) (SEQ ID No. 45), as shown in table 2; as shown in Table 3, it is a series of siRNA sequences designed for haplotype H2 (AGTGCCCAG) (SEQ ID NO. 46).
TABLE 2 siRNA sequences for haplotype H1 (CGTGCCCAT)
TABLE 3 siRNA sequences for haplotype H2 (CAGTGCCCAGG)
To verify the interfering efficiency of individual sirnas, 3 hepatocellular carcinoma cell lines were selected to represent three common genotypes: hepG2 (H1 type), huh-6 (heterozygous, comprising both H1 and H2 genotypes) and SNU387 (H2 type). 3 cells were transfected with the siRNAs in tables 2 and 3, respectively, as well as negative control siRNA (Ruibo organism) and positive control siRNA (Ruibo organism). Transfection Using Lipofectamine TM RNAiMAXP (Thermo Fisher Scientific) reagent, transfection procedure reverse transfection assays were performed according to Lipofectamine RNAiMAX recommended procedure. Cells were collected 24 hours after transfection and the interference efficiency of siRNA was detected by qRT-PCR. As shown in FIG. 5, different siRNAs have different interference efficiencies. SiRNA si1# and si2# targeted to H1 type have the best interference efficiency in HepG2 cells carrying H1, can inhibit 60% of gene expression, and the rest of Si5#, si6#, si7#, si8# and si11# can reach 40% of inhibition efficiency. si-1# and si-2# carry H1 and H2 in heterozygous formThe inhibition efficiency in Huh-6 was 50% and only about 20% in SNU387 carrying H2. siA6# in H2-targeting siRNAs inhibited gene expression by about 40% in SNU387, whereas inhibition was poor in HepG2 and Huh 6. The experimental results confirm the specific inhibition of siRNA expression on different genotypes.
Example 6:the siRNA targeting the region can effectively inhibit the expression of AURKB genes in tumor cells and inhibit the growth of tumors without affecting the growth of heterozygous cells
3 siRNAs (si1#, si2#, si6#) were selected for CellTiter-Glo (CTG) method growth experiments, and the effect of AURKB interference on the growth of each cell was observed. Test procedures cells were tested on days 1,2,3, and 4 after transfection, respectively, according to the methods recommended by CellTiter-Glo manual. As shown in fig. 6, si-1# and si-2# can significantly inhibit growth of HepG2 carrying H1 type, si-a6# significantly inhibited growth of cell line SNU387 carrying H2 type, but none of the three sirnas significantly affected cell growth in heterozygous cell line Huh-6. Experimental results show that the target of the patent description region can specifically inhibit the growth of tumors. For tumor cells with LOH, the siRNA can effectively inhibit the expression of AURKB genes and inhibit the growth of the tumor cells, and for heterozygous normal cells without LOH, the siRNA does not influence the normal growth of the heterozygous normal cells.
It should be understood that while the present invention has been described by way of example in terms of its preferred embodiments, it is not limited to the above embodiments, but is capable of numerous modifications and variations by those skilled in the art. The sequence transformation, nucleotide modification and the like of the siRNA targeting the AURKB gene H1/H2 haplotype can be correspondingly adjusted and changed according to specific needs. It will thus be appreciated that those skilled in the art will be able to devise numerous alternative arrangements which, although not explicitly described herein, embody the principles of the invention and are included within its spirit and scope.

Claims (34)

  1. Sirna targeting a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
  2. 2. siRNA according to claim 1, wherein at least one strand consists of a nucleotide chain of 19-30 nucleotides in length, preferably of a nucleotide chain of 19-29, 19-28, 19-27, 19-26, 19-25 nucleotides in length.
  3. 3. The siRNA of claim 1, wherein said siRNA comprises at least one modified nucleotide.
  4. 4. The siRNA of claim 1, wherein the 3' end of each strand of the siRNA is modified with a overhang base.
  5. 5. The siRNA of claim 4 wherein said overhang base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
  6. 6. The siRNA of claim 1, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO:1 and SEQ ID NO:2;
    SEQ ID NO:3 and SEQ ID NO:4, a step of;
    SEQ ID NO:5 and SEQ ID NO:6, preparing a base material;
    SEQ ID NO:7 and SEQ ID NO:8, 8;
    SEQ ID NO:9 and SEQ ID NO:10;
    SEQ ID NO:11 and SEQ ID NO:12;
    SEQ ID NO:13 and SEQ ID NO:14;
    SEQ ID NO:15 and SEQ ID NO:16;
    SEQ ID NO:17 and SEQ ID NO:18;
    SEQ ID NO:19 and SEQ ID NO:20, a step of;
    SEQ ID NO:21 and SEQ ID NO:22;
    SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base;
    SEQ ID NO:25 and SEQ ID NO:26;
    SEQ ID NO:27 and SEQ ID NO:28;
    SEQ ID NO:29 and SEQ ID NO:30;
    SEQ ID NO:31 and SEQ ID NO:32;
    SEQ ID NO:33 and SEQ ID NO:34;
    SEQ ID NO:35 and SEQ ID NO:36;
    SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process;
    SEQ ID NO:39 and SEQ ID NO:40, a step of performing a;
    SEQ ID NO:41 and SEQ ID NO:42; and
    SEQ ID NO:43 and SEQ ID NO:44.
  7. 7. a vector comprising the siRNA of any one of claims 1-6.
  8. 8. A cell comprising the siRNA of any one of claims 1-6.
  9. 9. A pharmaceutical composition for inhibiting tumor growth comprising the siRNA of any one of claims 1-6 and a pharmaceutically acceptable excipient.
  10. 10. The pharmaceutical composition of claim 9, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  11. 11. The composition of claim 9, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
  12. 12. A pharmaceutical composition for inhibiting tumor growth comprising an siRNA that inhibits AURKB expression and a pharmaceutically acceptable excipient.
  13. 13. The pharmaceutical composition of claim 12, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  14. 14. The composition of claim 13, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
  15. 15. A method of reducing AURKB expression in a target cell by administering the siRNA of any one of claims 1-6.
  16. 16. A method of inhibiting tumor growth by administering to a subject in need thereof an siRNA targeted by a polypeptide comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
  17. 17. The method according to claim 16, wherein at least one strand of said siRNA consists of a nucleotide strand of 19-30 nucleotides in length, preferably of 19-29, 19-28, 19-27, 19-26, 19-25 nucleotides in length.
  18. 18. The method of claim 16, wherein the siRNA comprises at least one modified nucleotide.
  19. 19. The method of claim 16, wherein the 3' end of each strand of the siRNA is modified with a overhanging base.
  20. 20. The method of claim 16, wherein the overhanging base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
  21. 21. The method of claim 16, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO:1 and SEQ ID NO:2;
    SEQ ID NO:3 and SEQ ID NO:4, a step of;
    SEQ ID NO:5 and SEQ ID NO:6, preparing a base material;
    SEQ ID NO:7 and SEQ ID NO:8, 8;
    SEQ ID NO:9 and SEQ ID NO:10;
    SEQ ID NO:11 and SEQ ID NO:12;
    SEQ ID NO:13 and SEQ ID NO:14;
    SEQ ID NO:15 and SEQ ID NO:16;
    SEQ ID NO:17 and SEQ ID NO:18;
    SEQ ID NO:19 and SEQ ID NO:20, a step of;
    SEQ ID NO:21 and SEQ ID NO:22;
    SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base;
    SEQ ID NO:25 and SEQ ID NO:26;
    SEQ ID NO:27 and SEQ ID NO:28;
    SEQ ID NO:29 and SEQ ID NO:30;
    SEQ ID NO:31 and SEQ ID NO:32;
    SEQ ID NO:33 and SEQ ID NO:34;
    SEQ ID NO:35 and SEQ ID NO:36;
    SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process;
    SEQ ID NO:39 and SEQ ID NO:40, a step of performing a;
    SEQ ID NO:41 and SEQ ID NO:42; and
    SEQ ID NO:43 and SEQ ID NO:44.
  22. 22. the method of claim 16, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  23. 23. The method of claim 22, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
  24. 24. A method of inhibiting tumor growth, the method comprising administering to a subject in need thereof an siRNA that inhibits AURKB expression.
  25. 25. The method of claim 24, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  26. 26. The method of claim 25, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
  27. 27. Use of an siRNA targeted by a polypeptide comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46, and a fragment of mRNA encoded by the sequence shown in SEQ ID NO. 46.
  28. 28. The use according to claim 27, wherein at least one strand of said siRNA consists of a nucleotide chain of 19 to 30 nucleotides in length, preferably of 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25 nucleotides in length.
  29. 29. The use of claim 27, wherein said siRNA comprises at least one modified nucleotide.
  30. 30. The use of claim 27, wherein the 3' end of each strand of said siRNA is modified with a overhang base.
  31. 31. The use of claim 27, wherein the overhanging base consists of any one or more deoxyribonucleotides in dA, dT, dG, dC.
  32. 32. The use of claim 27, wherein the siRNA has a structure selected from the group consisting of:
    SEQ ID NO:1 and SEQ ID NO:2;
    SEQ ID NO:3 and SEQ ID NO:4, a step of;
    SEQ ID NO:5 and SEQ ID NO:6, preparing a base material;
    SEQ ID NO:7 and SEQ ID NO:8, 8;
    SEQ ID NO:9 and SEQ ID NO:10;
    SEQ ID NO:11 and SEQ ID NO:12;
    SEQ ID NO:13 and SEQ ID NO:14;
    SEQ ID NO:15 and SEQ ID NO:16;
    SEQ ID NO:17 and SEQ ID NO:18;
    SEQ ID NO:19 and SEQ ID NO:20, a step of;
    SEQ ID NO:21 and SEQ ID NO:22;
    SEQ ID NO:23 and SEQ ID NO:24, a step of detecting the position of the base;
    SEQ ID NO:25 and SEQ ID NO:26;
    SEQ ID NO:27 and SEQ ID NO:28;
    SEQ ID NO:29 and SEQ ID NO:30;
    SEQ ID NO:31 and SEQ ID NO:32;
    SEQ ID NO:33 and SEQ ID NO:34;
    SEQ ID NO:35 and SEQ ID NO:36;
    SEQ ID NO:37 and SEQ ID NO:38, a step of carrying out the process;
    SEQ ID NO:39 and SEQ ID NO:40, a step of performing a;
    SEQ ID NO:41 and SEQ ID NO:42; and
    SEQ ID NO:43 and SEQ ID NO:44.
  33. 33. the use of claim 27, wherein the tumor is selected from the group consisting of: glioma, leukemia, brain cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, breast cancer, intestinal cancer, nasopharyngeal cancer, endometrial cancer, and prostate cancer.
  34. 34. The use of claim 33, wherein the tumor is selected from the group consisting of brain cancer, lung cancer, and liver cancer.
CN202211176786.3A 2022-09-26 2022-09-26 siRNA for inhibiting tumor growth and application thereof Pending CN117757789A (en)

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US7615627B2 (en) * 2006-06-21 2009-11-10 Reliance Life Sciences Pvt. Ltd. RNA interference mediated inhibition of aurorakinase B and its combinations as anticancer therapy
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