WO2019047368A1

WO2019047368A1 - Mirna targeting cjun signaling pathway and preparation method therefor and use thereof

Info

Publication number: WO2019047368A1
Application number: PCT/CN2017/111054
Authority: WO
Inventors: 钱政江; 李翔; 杨海洋
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2017-09-08
Filing date: 2017-11-15
Publication date: 2019-03-14
Also published as: CN107502609A

Abstract

Disclosed is an miRNA targeting the cJun signaling pathway and a preparation method therefor and the use thereof. In particular, the present invention relates to a micromolecule RNA-miR-4632 targeted inhibiting the cJun gene and the use thereof. The nucleotide sequence of the miRNA is SEQ ID NO. 1 or a nucleotide sequence having at least 90% identity thereto. The miRNA has an endogenous, specific targeted inhibition of the cJun signaling pathway and can be used to prepare a drug that inhibits diseases caused by abnormal activation of the cJun-mediated signaling pathway.

Description

miRNA targeting cJun signaling pathway and preparation method and application thereof

Technical field

The present invention relates to the field of biomedicine, in particular to a miRNA targeting a cJun signaling pathway and a preparation method and application thereof, in particular to a small molecule RNA-miR-4632 targeted for inhibiting cJun gene and application thereof, in particular to hsa- The use of miR-4632 in the preparation of a drug caused by a cJun-mediated abnormal activation of a signaling pathway.

Background technique

The cell proto-oncogene cJun is an important transcription factor in the nucleus and a member of the activation protein (AP-1) family; cJun participates in many genes in the form of a single transcription factor or AP-1. Transcriptional regulation, and thereby play various biological functions. After being stimulated by various external signals such as cytokines, growth signals, ultraviolet rays or environmental pollutants, the cells are induced to activate cJun via tyrosine kinase-related receptors and cJun amino terminal kinase JNK. A specific DNA fragment in the nucleus regulates transcription of the target gene. The cJun signaling pathway is involved in the regulation of cell proliferation, differentiation, cycle and apoptosis.

Studies have shown that cJun mediates the abnormal activation of signaling pathways, and the occurrence of cardiovascular and cerebrovascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension and atherosclerosis. Development related. In addition, the continuous activation of this signaling pathway is also closely related to the formation of gastrointestinal diseases, inflammatory diseases, prostate, nervous system diseases and malignant tumors (such as esophageal cancer, ovarian cancer). Therefore, the cJun signaling pathway has great potential as a new drug target for the treatment of related diseases.

However, there are currently relatively few specific inhibitors developed for the cJun signaling pathway. CN 104080469A discloses the use of novel JNK inhibitor molecules and their use in a method for treating human or animal body by treatment, which is mainly directed to the JJ upstream signal molecule JNK, which has poor biostability and availability. Low, complicated preparation process and other shortcomings.

Other specific inhibitors of the cJun signaling pathway include peptide and peptidomimetic inhibitors, natural product inhibitors, and small molecule inhibitors discovered by computer drug virtual screening techniques. (1) Peptide and peptidomimetic inhibitors are phosphopeptides designed by using the amino acid residue sequence of the related molecule phosphorylation product on the JNK signaling pathway as a template to block the transmission of JNK signaling to cJun to achieve inhibitory effects, but such inhibition The agent is easily metabolized in the body and has low bioavailability; (2) natural product inhibitors mainly include natural chemical products such as terpenoids and flavonoids, natural active molecules which inhibit the JNK signaling pathway, thereby inhibiting cJun Signal transmission, but the process of obtaining such biologically active compounds is complicated, the process is cumbersome, and the cJun signaling pathway cannot be specifically inhibited; (3) a series of inhibitory activities against JNK signaling pathway can be obtained by computer drug virtual screening technology. Small molecule compounds, but many screened small molecule compounds are difficult to synthesize and it is difficult to maintain high biosuppressive activity.

Therefore, the prior art has yet to be improved and developed, and it is necessary to develop a more effective cJun kinase signaling pathway inhibitor.

Summary of the invention

The following is an overview of the topics detailed in this document. This Summary is not intended to limit the scope of the claims.

In view of the deficiencies of the prior art, the present application provides a miRNA targeting a cJun signaling pathway and a preparation method and application thereof, which have endogenous and specific targeting inhibition cJun (proto-oncogene) signaling pathway, A drug capable of inhibiting a disease caused by abnormal activation of a cJun-mediated signaling pathway can be prepared.

To this end, the application uses the following technical solutions:

In a first aspect, the application provides a miRNA that targets a cJun signaling pathway, the nucleotide sequence of which is SEQ ID NO. 1 or a nucleotide sequence having at least 90% identity thereto.

The nucleotide sequence shown in SEQ ID NO. 1 is as follows: 5'-UGCCGCCCUCUCGCUGCUCUAG-3'.

According to the present application, the nucleotide sequence of the miRNA is the nucleotide sequence shown in SEQ ID NO.

In the present application, by overexpressing the miRNA of the present application, i.e., miR-4632, in human pulmonary artery smooth muscle cells, the activation or expression of cJun signaling pathway by platelet-derived growth factor BB (PDGFBB) can be inhibited; Application of miRNA, miR-4632, inhibits platelet-derived growth factor BB (PDGFBB)-activated cJun signaling pathway-induced abnormal proliferation of human pulmonary artery smooth muscle cells.

In the present application, the preparation method of the miRNA includes a method for whole-gene synthesis and clonal expression of a mature sequence, and the method for expressing the clone is a conventional technique in the art, and a person skilled in the art can construct according to a suitable method as needed. This is not particularly limited.

In a specific embodiment, the method of constructing the miRNA comprises the following steps:

(1) Designing primers and performing PCR amplification using human genomic DNA as a template;

(2) inserting the PCR amplified fragment between XhoI and EcoRI of the plv4/EGFP lentiviral vector;

(3) Transfecting the constructed lentiviral vector into cells to express miRNA.

The nucleotide sequence of the primer is shown in SEQ ID NO. 2-3;

The nucleotide sequence shown in SEQ ID NO. 2 is as follows: 5'-CCGCTCGAGGACGAGCAGGACTGCGGA-3';

The nucleotide sequence shown in SEQ ID NO. 3 is as follows: 5'-CCGGAATTCCAAGGACCTGAGCCCCAC-3'.

In a second aspect, the application provides the use of a miRNA according to the first aspect, in the manufacture of a medicament or kit for the diagnosis, prevention, treatment or prognosis of a disease.

According to the present application, the disease is a disease caused by abnormal activation of a cJun-mediated signaling pathway.

According to the present application, the disease is any one or a combination of at least two of cardiovascular and cerebrovascular diseases, gastrointestinal diseases, inflammatory diseases, leukemias, prostate diseases, nervous system diseases, tumors or pulmonary hypertension.

In the present application, the cardiovascular and cerebrovascular diseases include cardio-cerebral vascular diseases such as cardiac hypertrophy, cerebral ischemia-reperfusion injury, heart failure, myocardial infarction, ischemia reperfusion, hypertension, and atherosclerosis; Malignant tumors such as esophageal cancer or ovarian cancer.

In the present application, "cerebral ischemia-reperfusion" refers to ligating the left anterior descending branch of the coronary artery to cause myocardial ischemia for a period of time, and then releasing the ligature to resuspend the myocardium.

In a third aspect, the application provides a lentiviral vector comprising the nucleotide sequence of the miRNA of the first aspect.

In a fourth aspect, the present application provides a recombinant lentivirus comprising a recombinant lentivirus obtained by co-transfecting a lentiviral vector according to the third aspect with a packaging helper plasmid into a mammalian cell;

Preferably, the mammalian cell is a HEK293T cell.

In a fifth aspect, the present application provides a pharmaceutical composition comprising the miRNA of the first aspect.

According to the present application, the effective dose of the miRNA is 10-200 mg/kg, for example, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/ Kg, 90 mg/kg, 100 mg/kg, 120 mg/kg, 130 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg or 200 mg/kg.

According to the present application, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable virus, carrier or adjuvant.

In the present application, a pharmaceutically acceptable virus, carrier or adjuvant is selected from the group consisting of chitosan, cholesterol, liposomes or nanoparticles.

In a sixth aspect, the present application provides a kit for the diagnosis and/or prognosis of a disease, the kit comprising the miRNA of the first aspect and/or a probe or primer for specifically detecting the miRNA.

According to the application, the nucleotide sequence of the probe or primer is as shown in SEQ ID NO. 2-3;

Compared with the prior art, the present application has the following beneficial effects:

(1) This application finds that small miRNAs target cJun and inhibit the activity of this signaling pathway by inhibiting the expression of cJun molecules. It can be used as a novel cJun kinase inhibitor for the treatment of abnormal activation of cJun kinase signaling pathway. Disease

(2) The miRNA of the present application is a human endogenous miRNA, which is less toxic to the human body and can be better utilized by the human body, and can be introduced into the human body by intravenous injection and transported to a specific site through blood circulation, thereby achieving therapeutic purposes;

(3) The miRNA of the present application can be used as a novel cJun kinase signaling pathway inhibitor for the treatment of cardiovascular diseases, gastrointestinal diseases, inflammatory diseases, prostate, nervous system diseases and malignant tumors.

DRAWINGS

Figure 1A shows the 3'-UTR binding site of miR-4632 and cJ Jun;

Figure 1B shows the effect of overexpression of miR-4632 on cJun-UTR or cJun-UTR-mut luciferase activity, wherein cJun-UTR-mut is a mutant vector constructed based on the binding site for 3'-UTR;

Figure 1C shows the effect of overexpression of miR-4632 on the expression of cJun total protein in human pulmonary artery smooth muscle cells, wherein Mimic Ctrl is a chemically synthesized miR-4632 analog;

Figure 1D shows the effect of overexpression of miR-4632 on phosphorylation-activated cJun protein expression after PDGFBB stimulation of human pulmonary artery smooth muscle cells. Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence. Mimic Ctrl-1 is not treated with PDGF. , Mimic Ctrl-2 is processed with PDGF;

Figure 2A is a typical fluorescent picture of the effect of overexpression of miR-4632 on cell proliferation after PDGFB stimulates human pulmonary artery smooth muscle cells by EdU. Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence. Mimic Ctrl-1 does not use PDGF. Processing, Mimic Ctrl-2 is processed with PDGF;

Figure 2B is a statistical result of the effect of overexpression of miR-4632 on cell proliferation after PDGFB stimulates human pulmonary artery smooth muscle cells by EdU. Mimic Ctrl is a structurally similar but meaningless sequence of chemical synthesis, and Mimic Ctrl-1 is not treated with PDGF. , Mimic Ctrl-2 is processed with PDGF;

Figure 2C shows the effect of overexpression of miR-4632 on PCNA protein expression after PDGFBB stimulates human pulmonary artery smooth muscle cells. Mimic Ctrl is a chemically synthesized structurally similar but meaningless sequence. Mimic Ctrl-1 is not treated with PDGF, Mimic Ctrl -2 was treated with PDGF.

Detailed ways

The technical solutions of the present application are further described in the following with reference to the accompanying drawings, and the present invention is not limited to the scope of the embodiments.

Unless otherwise stated, the various starting materials used in the following examples were all commercially available and the methods employed were conventional techniques.

Example 1 Preparation of miRNA miR-4632

The mature sequence of miR-4632 is 5'-UGCCGCCCUCUCGCUGCUCUAG-3' (22 bp, SEQ ID NO. 1) can be obtained by:

1) Chemical synthesis

The analog of miR-4632 (miR-4632mimic) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. It is a double-stranded RNA that mimics high-level expression of endogenous mature miR-4632; standard purification, stored at -20 °C. The 20 M storage solution was dissolved in RNase-free water, placed at -80 ° C until use, and diluted to the desired concentration during use.

2) Viral vector-mediated expression of miR-4632

(1) The mature sequence and precursor sequence (pre-miRNA) information of miR-4632 was obtained in the miRBase (http://www.mirbase.org/) database. Two flanking sequences of about 300 bp in the mature sequence were found in the human genome database. Primers were designed based on the flanking sequences, and the restriction sites and protected bases of XhoI and EcoRI were added respectively to design primers:

Upstream primer 5'-CCGCTCGAGGACGAGC AGGACTGCGGA-3' (SEQ ID NO. 2);

The sequence of the downstream primer is 5'-CCGGAATTCCAAGGACCTGAGCCC CAC-3' (SEQ ID NO. 3);

(2) Using human genomic DNA as a template, PCR amplification was carried out by designing primers. The PCR reaction conditions were: denaturation at 95 ° C for 2 minutes, 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds for 35 cycles, 72 °C extension for 3 minutes;

(3) After amplifying the amplified PCR product agarose gel, the fragment was recovered by EZNA Gel Extraction Kit (Omgega), and digested with restriction endonucleases XhoI and EcoRI (NEB). The digested PCR fragment was recovered as an insert and inserted into the PLV4/EGFP lentiviral vector according to the DNA purification kit (EZNA Cycle-Pure Kit, Omgega); the vector was restricted with restriction enzymes XhoI and EcoRI (NEB). After double digestion, recovery and purification as a vector fragment;

(4) The insert and the vector fragment were ligated with T4 ligase (Promega) for 2 h at 16 ° C, then the ligated product was transformed into E. coli competent STBL3, and cultured at 37 ° C overnight to pick good growth. The single colony is amplified in LB medium containing the corresponding antibiotic of the vector, and the plasmid is extracted and verified by sequencing;

(5) The prepared DNA vector can be transfected into cells to transiently express miRNA, ie miR-4632.

The resulting lentiviral vector can be used for lentiviral packaging, as described below:

(1 ') The cell line required for lentiviral packaging is the HEK293T cell line, and the desired plasmid includes the lentiviral expression vector pLVX-miR-4632 and a lentiviral packaging plasmid. Cell transfection was carried out by conventional calcium phosphate transfection method, mixed with 2M CaCl ₂ and plasmid, then added dropwise to 2×HBS solution and gently shaken, and finally the mixed solution was slowly and uniformly added to the cell culture solution. Change the culture solution after 12 hours of transfection;

(2') After 48-72 hours of transfection, the cell culture medium containing the virus particles was collected, centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was collected and stored at -80 ° C for use;

(3') The packaged lentivirus can be used to infect selected cells, and cells stably stabilizing miR-4632 can be obtained by screening with the antibiotic puromycin.

Example 2 miR-4632 Targets Inhibition of cJun Molecules and Their Signaling Pathways

(1) Cell culture: HEK293A cells (purchased from ATCC, Manassas, VA) were cultured in DMEN medium containing 10% fetal bovine serum, and cells grown in good condition were collected, and seeded in a 24-well plate at 6 × 10 ⁴ /well. Internally cultured at 37 ° C, 5% CO _{2 for} 24 hours;

(2) Human pulmonary artery smooth muscle cells were purchased from Lonza (Walkersville, MD), cultured in SMCM complete medium containing 5% serum, and cells grown in good condition were collected, centrifuged, and inoculated into a 60 mm dish at 6×10 ⁵ . Incubate at 37 ° C, 5% CO _{2 for} 24 hours;

(3) Transfection: cells were transfected with calcium phosphate method when the cell density reached 70%, and co-transfected with CCJUN's 3'-UTR dual luciferase reporter vector or mutant 3'-UTR dual luciferase reporter vector. miR-4632 overexpression vector pLVX-CMV-miR-4632 or pLVX-CMV-control, after two days of transfection, the culture solution was removed, washed twice with PBS, and then lysed with 50 μL of 1× lysate;

(4) The chemically synthesized miR-4632 analog (mimic) was used to overexpress or inhibit the expression of miR-4632, and cel-miR-67 was used as a negative control, all purchased from Guangzhou Ruibo Bio. The cells were seeded in a Petri dish. When the cell density reached 70%, transfection was performed by Lipofectamine 2000 (Invitrogen), the medium was changed after 6 hours of transfection, and the culture was continued for 24 hours for the experiment;

Luciferase assay

The luciferase activity assay was performed using the dual luciferase assay kit (E1810, Promega) reference specification, and the corrected luciferase activity value was obtained by dividing the firefly luciferase value by the internal reference renilla luciferase reading. The luciferase activity values of the 3'-UTR vector of CCJUN were compared with the control group.

The 3'-UTR binding site of the miR-4632 and cJun is shown in FIG. 1A, and the fluorescein detection result is shown in FIG. 1B. Compared with the control, the CJUN luciferase activity of the overexpressing miR-4632 group is remarkable. The decrease indicates that miR-4632 may bind to the 3'-UTR of cJun; the mutation vector cJun-UTR-mut of 3'-UTR is constructed based on the binding site, and the dual luciferase activity is detected again by using the mutant vector. Mutation of the cJun binding site results in restoration of luciferase activity compared to wild-type UTR.

Protein hybridization assay

The total protein of the cells was collected, and the protein was quantified and subjected to polyacrylamide gel electrophoresis. The cJun protein antibody was used to detect cJun expression. As shown in Fig. 1C, overexpression of miR-4632 inhibited endogenous human pulmonary artery smooth muscle cells compared with the control. Expression of cJun; detection of phosphorylated cJun by cJun phosphorylation antibody, compared with control (Ctrl-1 for PDGF untreated indicates normal cell function, Ctrl-2 for PDGF treatment indicates abnormal cell function), as shown in Figure 1D Overexpression of miR-4632 significantly inhibited phosphorylation of cJun.

The results of this example demonstrate that miR-4632 inhibits CJUN expression and the CJUN kinase signaling pathway by targeting the 3'-UTR of CJUN.

Example 3 miR-4632 inhibits platelet-derived growth factor BB (PDGFBB)-induced proliferation of human pulmonary artery smooth muscle cells.

To investigate the role of miR-4632 in the activation of cJun signaling pathway by platelet-derived growth factor BB (PDGFBB), which leads to the regulation of dysfunction of human pulmonary artery smooth muscle cells: lentivirus infection with miR-4632 to increase miR- in human pulmonary artery smooth muscle cells The level of 4632 was then determined by cell proliferation marker protein PCNA and EdU cell proliferation assay to determine the level of cell proliferation.

The EdU cell proliferation assay kit (Ribo Biotechnology) was used for the EdU labeling, and the experimental procedure was carried out according to the kit instructions. The main steps were as follows: 1) Cell transfection: 1×10 ⁴ cells were seeded in a 48-well plate and cultured for 24 hours. Transfection experiments, after 24 hours of culture on the second day, starved with serum-free medium, then platelet-derived growth factor BB (PDGFBB) and 20 μM EdU were continued for 4 hours; 2) Cell staining was taken: cells were treated with 4% paraformaldehyde After fixing for 30 minutes at room temperature, 0.5% Triton X-100 was permeabilized for 10 minutes, washed with PBS, and then incubated with 150 μL of 1×Apollo staining solution for 30 minutes per well. The DNA was stained with 1×Hochest (150 μL per well) for 5 minutes. Photographed under a fluorescence microscope.

The results are shown in Fig. 2A, Fig. 2B and Fig. 2C. It can be seen from Fig. 2A and Fig. 2B that, under the stimulation of PDGFBB, compared with the control (Ctrl-1 is PDGF untreated, indicating that the cell function is normal, Ctrl-2 is PDGF treatment). Overexpression of miR-4632 reduced the proliferation of human pulmonary artery smooth muscle cells by 32%; from Fig. 2C, overexpression of miR-4632 also down-regulated the expression of PCNA of human pulmonary artery smooth muscle proliferation marker protein by 31%; 4632 has a significant inhibitory effect on the proliferation of pulmonary artery smooth muscle cells induced by growth factors.

In summary, the present application found that small miRNAs targeting cJun inhibit the activity of this signaling pathway by inhibiting the expression of cJun molecules, and can be used as a novel cJun kinase inhibitor for the treatment of abnormal activation by the cJun kinase signaling pathway. The disease caused.

The applicant claims that the present application illustrates the detailed method of the present application by the above embodiments, but the present application It is not limited to the above detailed method, that is, it does not mean that the present application must rely on the detailed method described above to be implemented. It should be apparent to those skilled in the art that any modification of the present application, the equivalent replacement of each raw material of the product of the present application, the addition of an auxiliary component, the selection of a specific manner, and the like, are all within the scope of protection and disclosure of the present application.

Claims

A miRNA that targets the cJun signaling pathway, the nucleotide sequence of which is SEQ ID NO. 1 or a nucleotide sequence having at least 90% identity thereto.
The miRNA according to claim 1, which has the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.
Use of the miRNA according to claim 1 or 2 for the preparation of a medicament or kit for the diagnosis, prevention, treatment or prognosis of a disease.
The use according to claim 3, wherein the disease is a disease caused by abnormal activation of a cJun-mediated signaling pathway.
The use according to claim 4, wherein the disease is any one or at least two of cardiovascular and cerebrovascular diseases, gastrointestinal diseases, inflammatory diseases, leukemias, prostate diseases, nervous system diseases, tumors or pulmonary hypertension Combination of species.
A lentiviral vector comprising the nucleotide sequence of the miRNA of claim 1 or 2.
A recombinant lentivirus obtained by co-transfecting a lentiviral vector according to claim 6 with a packaging helper plasmid into a mammalian cell.
The recombinant lentivirus according to claim 7, wherein the mammalian cell is a HEK293T cell.
A pharmaceutical composition comprising the miRNA of claim 1 or 2.
The pharmaceutical composition according to claim 9, wherein the miRNA has an effective dose of 10 to 200 mg/kg.
The pharmaceutical composition according to claim 9, which further comprises any one or a combination of at least two of a pharmaceutically acceptable virus, carrier or adjuvant.
A kit for diagnosing and/or prognosing a disease, comprising the claim 1 or 2 The miRNA and/or probe or primer for specifically detecting the miRNA.
The kit according to claim 12, wherein the nucleotide sequence of the probe or primer is as shown in SEQ ID NO. 2-3.