CN109394779B

CN109394779B - miR-183 regulator of liver cells and application of miR-183 regulator in preparation of medicine for treating and regulating liver cells

Info

Publication number: CN109394779B
Application number: CN201811209764.6A
Authority: CN
Inventors: 靳伟; 张瑞芝; 张春艳; 郭建林; 常翠芳; 石金保; 徐存拴
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2018-10-17
Filing date: 2018-10-17
Publication date: 2021-11-02
Anticipated expiration: 2038-10-17
Also published as: CN109394779A

Abstract

The invention relates to an application of a miR-183 regulator for regulating liver cells in preparing a medicament for treating and regulating liver cells, wherein the regulator comprises a regulator for inhibiting the expression of Tnfrsf1 alpha and/or promoting the expression of a protein related to liver cell proliferation; a liver cell modulating drug is a drug that contains the modulator to enable the modulator to function. Modulators include miR-183 mimetics that are capable of promoting liver cell proliferation. Modulators also include inhibitors that promote apoptosis of liver cells. The invention provides a regulator capable of regulating liver cells and application thereof in preparing a medicament for treating and regulating liver cell proliferation, and the applicant discovers through research that the regulator capable of regulating liver cell proliferation or apoptosis, namely miR-183 simulant and inhibitor respectively have obvious effects of promoting liver cell proliferation and inhibiting liver cell proliferation, so that the regulator can be prepared into corresponding medicaments to regulate liver cells.

Description

miR-183 regulator of liver cells and application of miR-183 regulator in preparation of medicine for treating and regulating liver cells

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a miR-183mimic capable of regulating liver cells, an inhibitor, a recombinant expression vector of the inhibitor and application of the recombinant expression vector.

Background

The liver is an important organ of the body and has the functions of storage, metabolism, biotransformation, detoxification, hematopoiesis, bile pigment synthesis, secretion, regeneration and the like. The research on the effect of the liver regeneration related gene on hepatocyte proliferation and liver regeneration has important theoretical significance and application value for disclosing the liver regeneration mechanism, constructing artificial liver, establishing the method for treating and preventing liver diseases and the like.

microRNA (miRNA) is a non-coding single-stranded RNA molecule with the length of about 22 nucleotides and coded by endogenous genes, and 28645 miRNA molecules have been found in animals, plants and viruses to date. The miRNA recognizes and connects with the 3'UTR of mRNA transcribed by a target gene, binds to a specific recognition region on the 5' UTR through a specific binding site, and then forms a silencing complex with a series of functional proteins to induce the degradation of the mRNA, thereby blocking the translation of the target gene and the expression of the proteins at the post-transcriptional level. According to the function difference of target genes, miRNA can participate in cell cycle, differentiation and apoptosis, and is increasingly paid high attention by researchers.

miR-183 is one of miR-183 families (including miR-183, miR-182 and miR-96), and researches show that miR-183 has higher expression in tumor tissues such as colorectal cancer and prostate cancer than tumor surrounding tissues, and can be used as one of indexes for early diagnosis and prognosis evaluation of tumors. The expression level of miR-183 in liver cirrhosis, precancerous lesion and liver cancer tissues is higher than that of a healthy control group, and the miR-183 participates in the occurrence and development process of liver cancer by inhibiting the expression of a target gene AKAP12 gene. Various bioinformatics software predicts that miR-183 can regulate the expression of multiple target genes such as MTA1, Tnfrfsf 1 alpha, PDCD4 and AKAP 12. Among them, the encoded product of Tnfrsf1 alpha gene is the main receptor for TNF-alpha to induce hepatocyte apoptosis. When various injuries are caused, TNFRI is abundantly expressed on the liver cell membrane, and Tnfrfsf 1 alpha is in a key position in the process of TNF-alpha induced acute liver failure.

Disclosure of Invention

The invention provides a miR-183 regulator of liver cells and application thereof in preparing a medicament for treating and regulating the liver cells, wherein the stimulant and the inhibitor can be used for regulating the expression of Tnfrsf1 alpha protein, and realizing the research of basic medicine and clinical medicine for regulating the proliferation or apoptosis of the liver cells, thereby being beneficial to providing an efficient and high-flux screening and evaluating platform and means for developing related medicaments and greatly promoting the application of miRNA in the prevention, diagnosis and treatment of tumors.

The purpose of the invention is realized by the following technical scheme:

the application of a miR-183 regulator of liver cells in preparing a medicament for treating and regulating the liver cells, wherein the regulator comprises a regulator for inhibiting the expression of Tnfrsf1 alpha and/or promoting the expression of a protein related to liver cell proliferation; the liver cell regulating drug is a drug containing the regulator so that the regulator can play a role in promoting and/or inhibiting liver cell proliferation.

Further, the regulator comprises a miR-183mimic capable of promoting liver cell proliferation, and the base sequence of the miR-183mimic is as follows: SEQ ID NO.1, which SEQ ID NO. 1: uauggcacug guagaauuca cu are provided.

Further, the regulator also comprises an inhibitor capable of promoting liver cell apoptosis, and the base sequence of the inhibitor is as follows: SEQ ID No.2, wherein SEQ ID No.2 is: ggccacaccc ccacctcagg aacgggactc gaaggaccat cctgctagat gccctgcttc cctgtgaacc tcctctttgg tcctctaggg ggcaggctcg atctggcagg ctcgatctgg cagccacttc cttggtgcta ccgacttggt gtacatagct tttcccagct gccgaggaca gcctgtgcca gccacttgtg catggcaggg aagtgtgcca tctgctccca gacagctgag ggtgccaaaa gccaggagag gtgattgtgg agaaaaagca caatctatct gatacccact tgggatgcaa ggacccaaac aaagcttctc agggcctcct cagttgattt ctgggccctt ttcacagtag ataaaacagt ctttgtattg attatatcac actaatggat gaacggttga actccctaag gtaggggcaa gcacagaaca gtggggtctc cagctggagc ccccgactctTgtaaa taca ctaaaaatct aaaagtg are provided.

Further, the inhibitor is an inhibitor capable of binding with the miR-183mimic, and the binding site of the inhibitor and the miR-183mimic is GTGCCATC; if the two are combined through the sequence sites, the action of the miR-183mimic can be effectively inhibited, so that the action of Tnfrsf1 alpha for promoting the liver cell apoptosis is released, and the aim of promoting the liver cell apoptosis is fulfilled.

Furthermore, restriction enzyme sites, which generate sticky ends after enzyme digestion, are added to both the 5 'end and the 3' end in the base sequence SEQ ID NO.2 of the inhibitor;

further, the restriction enzyme cutting sites are nucleotide sequences corresponding to Xho I and Not I respectively; the XhoI cleavage site is located at the 5 'end, and the Not I cleavage site is located at the 3' end.

Further, the regulator also comprises a recombinant expression vector capable of promoting liver cell apoptosis, and the inhibitor is contained in the recombinant expression vector.

Further, the marker gene in the recombinant expression vector is dual luciferase.

Further, the recombinant expression vector is obtained by connecting the miR-183 inhibitor to a psi-CHECK-2 (namely psi-CHECKM-2) vector.

A modulator capable of modulating liver cells, said modulator being as described above.

The application of a modulator capable of regulating liver cells in promoting and inhibiting liver cell proliferation, wherein the modulator is the modulator described in any one of the above, and the modulator comprises a miR-183mimic capable of promoting liver cell proliferation, an inhibitor capable of inhibiting liver cell proliferation or a recombinant expression vector capable of inhibiting liver cell proliferation, and the application is specifically referred to the above definition.

Compared with the prior art, the invention has at least the following advantages:

the invention provides a miR-183 regulator of liver cells and application thereof in preparing medicaments for treating and regulating the liver cells, and the applicant surprisingly discovers regulators capable of regulating the proliferation or apoptosis of the liver cells, namely a miR-183 stimulant and an inhibitor, which respectively have obvious effects of promoting the proliferation of the liver cells and inhibiting the proliferation of the liver cells, so that the miR-183 stimulant and the inhibitor can be prepared into corresponding medicaments to regulate the liver cells.

The miR-183mimic can be used for reducing the expression of Tnfrsf1 alpha protein so as to promote the proliferation of liver cells, therefore, the miR-183mimic can be applied to the research of basic medicine and clinical medicine for promoting the proliferation of liver cells, and can also be further prepared into medicines for realizing the purpose of promoting the proliferation of liver cells. The inhibitor and the recombinant expression vector thereof can be used for inhibiting miR-183 and increasing the expression of Tnfrfsf 1 alpha protein, so that the apoptosis of liver cells is promoted, and therefore, the inhibitor can be applied to the research of basic medicine and clinical medicine for promoting the apoptosis of the liver cells, and can also be further prepared into medicines for realizing the regulation of the liver cells. The two methods are beneficial to providing an efficient and high-flux screening and evaluating platform and means for developing related drugs, and can greatly promote the application of miRNA in tumor prevention, diagnosis and treatment.

Drawings

FIG. 1 is a comparison of the high throughput assay results for miR-183 and the qRT-PCR assay results;

FIG. 2 is a graph showing that the MTT method is used for detecting the influence of mics and inhibitors of miR-183 on the proliferation activity of BRL-3A cells;

FIG. 3A shows that the EdU method detects the influence of mics and inhibitors of miR-183 on the proliferation activity of BRL-3A cells;

FIG. 3b is a result of statistical analysis of the proliferation activity of the miR-183mimic and inhibitor on BRL-3A cells;

FIG. 4 is a graph showing the effect of mic and inhibitor of miR-183 on the BRL-3A cell cycle;

FIG. 5 is a dual luciferase assay demonstrating the binding of miR-183 to the 3' -UTR of Tnfrsf1 α.

Detailed Description

The present invention will be further described with reference to the following drawings and examples, which are illustrative only and not intended to be limiting, and the scope of the present invention is not limited thereby.

Example 1: preparation and material drawing of rat liver regeneration model

The experimental rats were adult healthy male Sprague-Dawley rats weighing 230. + -.20 g, provided by the Experimental animals center of university of south river, university. The breeding temperature is 21 + -2 deg.C, relative humidity is 60 + -10%, illumination time is 12h/d (8:00-20:00), and drinking water and food intake are free. 114 of the rats were randomly divided into 19 groups of 6 rats each. Among them, normal control (0h)1 group, 2/3 liver resection (PH) and Sham Operation (SO) 9 groups. The pH group was performed by Higgins and Anderson, and the SO group was at the same pH except that liver lobes were not excised. At 2, 6, 12, 24, 30, 36, 72, 120 and 168h after surgery, the right lobe of the liver was placed in tissue storage reagent (e.g., RNAlater) and stored at-20 ℃ for use.

Example 2: high throughput sequencing of miRNAs

An appropriate amount of the above liver tissue stored at-20 ℃ was ground in a mortar containing liquid nitrogen, and mirRNAs were extracted and purified according to the Kit (mirVana mirNaAIassociation Kit, Ambion, USA) protocol. Total RNA quality was checked by agarose gel electrophoresis (180V, 0.5h), 28S rRNA:18S rRNA was about 2:1, and OD was determined separately₂₆₀And OD₂₈₀At OD₂₆₀/OD₂₈₀RNA is qualified if the RNA content is more than or equal to 2.0. Qualitative and quantitative analysis of miRNAs was performed by Shanghai Bohao Biotech, Inc., and the sequencing method was single-ended Solexa microRNA-Seq sequencing with a read length of 36 nt. And comparing the sequence of the miRNAs obtained by sequencing with a miRNAs library to determine the species and abundance of the miRNAs.

Example 3: rat liver regeneration related miRNAs

425 miRNAs are detected from 10 time points of rat regenerated livers of 0, 2, 6, 12, 24, 30, 36, 72, 120 and 168h in an experimental group (PH) and a pseudo-operation group (SO) by a single-ended Solexa microRNA-Seq high-throughput sequencing method, and ratio value analysis shows that 39 of 126 miRNAs with signal values larger than 20 have meaningful expression changes. Of these, 31 were considered to have a significant expression up-regulation, with the ratio value of 2-fold higher than that of the control. The ratio values of 4 strips were ≦ 2-fold for the control, and considered to be significant expression downregulation. 4 were up-regulated at some time points and down-regulated at some time points, considered up/down regulation. T-test showed that 23 of the 39 miRNAs with significant expression changes were associated with liver regeneration (Table 1).

TABLE 1 miRNAs with meaningful transcriptional changes in rat liver regeneration

Note:

means that the ratio value is greater than or equal to 2;

represents a ratio value of 0.5 or less;

representing P <0.05, presumably rat liver regeneration-associated microRNAs.

Example 4: real-time fluorescent quantitative PCR (qRT-PCR) detection

Designing a specific Stem-Loop Reverse Transcription (RT) primer (SEQ ID NO.3), a qRT-PCR upstream and downstream primer sequence and a U6 internal reference primer sequence, wherein the qRT-PCR primer comprises a miR-183 upstream primer (SEQ ID NO. 4) and a miR-183 downstream primer (SEQ ID NO.5), the U6 internal reference primer sequence comprises a U6 upstream primer (SEQ ID NO.6) and a U6 downstream primer (SEQ ID NO.7), performing species comparison with miRBase, and determining the specificity of the primers, wherein the sequences of the primers are shown in Table 2.

Total RNA extraction from cells was performed according to the instructions for RNA extraction reagent (Trizol) (Invitrogen, USA), and OD was measured with a spectrophotometer₂₆₀And OD₂₈₀At OD₂₆₀/OD₂₈₀The RNA is qualified when the ratio of 28S rRNA to 18S rRNA is about 2:1 when the ratio is more than or equal to 2.0 and the detection is carried out by denaturing agarose gel electrophoresis (70V,20 min). Using 2. mu.g of RNA as a template, reverse transcription was carried out according to the instructions of AMV reverse transcription kit (Promega, USA) to obtain first strand cDNA. Next, 1. mu.l of cDNA was taken, and 10. mu.l of a fluorescent dye mixture (SyBr Green I Mix), 0.4. mu.l of a primer, and 8.6. mu.l of purified water with an enucleating enzyme were added thereto. Mixing, amplifying Gene in a fluorescent quantitative PCR instrument (Rotor-Gene 3000) (Corbett Robotics, Australia), and detectingThe fluorescence signal value of the amplified product was compared with that of beta-actin (NM-031144) and the relative expression level (ratio value) of the gene was calculated as an internal reference. The conditions for qRT-PCR were all: 95 ℃ for 2min, 95 ℃ for 15sec, 60 ℃ for 20sec, 72 ℃ for 20sec, 40 cycles. Each sample was tested in triplicate and the data obtained was 2^-ΔΔCtThe method is used for relative quantitative treatment.

TABLE 2 reverse transcription primers and PCR primer sequences

Note: RT. reverse transcription primer; FP. an upstream primer; RP. downstream primer.

The expression change of miR-183 in the rat regenerated liver is verified by qRT-PCR, the verification result is shown in figure 1, and the expression trend of miR-183 is basically consistent with the high-throughput sequencing result.

Example 5: rat hepatocyte culture

The rat normal liver cell BRL-3A used in the experiment is purchased from cell resource center of Shanghai Life science research institute of Chinese academy of sciences, the culture medium is DMEM high-sugar medium containing 10% Fetal Bovine Serum (FBS), and the cell culture is performed at 37 deg.C, saturation humidity and 5% CO₂In the incubator. Cells in logarithmic growth phase were passaged at 5X 10⁴Individual cells/vial.

Example 6: MTT method for detecting proliferation of liver cells treated by regulator of the invention

The logarithmic growth phase of BRL-3A cells was digested with 0.25% pancreatic enzyme (Invitrogen, USA) at 1 ml/well and 5X 10⁴The cell suspension of each/ml cell was seeded in a 24-well cell culture plate, and after 12 hours of culture, cell transfection was performed according to the instructions for Lipofectamine RNAiMAX, Invitrogen, USA. Briefly, miR-183mimic (miR-183 mimic) (100nM), mimic NC (mimic negative control) (150nM), inhibitor (inhibitor) (100nM), inhibitor NC (inhibitor negative control) (150nM) and 1.5. mu.l of the transfection reagent were added to 25. mu.l of OPTI-MEM medium, respectively, and left to stand at room temperature for 5 min. Mixing the above solutions gently to form transfection complex, standing at room temperature for 20min, adding into a solution containing 0.45ml of OPTI-MEM medium (Gibco, USA) was incubated at 37 ℃ for 4 hours, and the medium was changed to normal medium. The experiment was repeated 3 times, with 3 replicate wells per experimental group.

Mu.l/well of MTT (Geneview, USA) was added to a 96-well plate containing cells and medium to a final concentration of 0.5g/L, and the plate was incubated at 37 ℃ for 4 hours in the dark, and after completely discarding the culture medium, 100. mu.l of dimethyl sulfoxide (DMSO, Geneview, USA) was added to each well and gently shaken for 10min to dissolve formazan crystals sufficiently. Finally, the absorbance of each well was measured at 490nm using a microplate reader (Biotek, USA). The experiment was repeated 3 times, with 3 replicate wells per experimental group.

The detection result is shown in fig. 2, the cell proliferation activity of the miR-183mimic treatment group is higher than that of the mimic NC group, and the miR-183mimic promotes the proliferation of BRL-3A cells; the cell proliferation activity of the inhibitor treated group is lower than that of the inhibitor NC group, which indicates that the inhibitor inhibits BRL-3A cell proliferation (P < 0.05).

Example 7: EdU labeling method for detecting hepatocyte proliferation after being treated by regulator of the invention

Taking BRL-3A cells in logarithmic growth phase at 5 × 10⁴The cell density of each well is inoculated in a 24-well plate of a preset round glass slide, after 12 hours of culture, mimic (100nM) and inhibitor (150nM) transfect BRL-3A cells cultured in vitro, after 48 hours of transfection, a cell slide is taken out, 2 hours before material drawing, EdU (Sharpbo, Guangzhou) is added to ensure that the final concentration is 50 mu M, and 4% paraformaldehyde is fixed for 30 minutes. Then, the cells were decolorized and incubated in glycine solution (2g/L) for 5 min. Then, the cells were decolorized and incubated in 0.5% TritonX-100 for 10 min. Then, the cells were incubated in 1 XApollo staining reaction (Sharpo, Guangzhou) for 30min, 0.5% TritonX-100 for 10-30min, and 1 Xhoechst 33342 reaction (Sharpo, Guangzhou) for 30min to mark the nuclei, each of which was washed 3 times with PBS for 5 min. Finally, 5 non-overlapping fields (20 ×) were randomly selected under the fluorescence microscope for observation and photography, and EdU positive cells and nuclei in the corresponding fields were counted separately using Image-Pro Plus 6.0 software. And analyzed for differences between groups using the one-way anova method of SPSS 13.0 statistical software.

The test results showed that the fraction of EdU-positive cells in the mimic group was 37.5%, which was significantly higher than that in the control group (30.5%) (P <0.05), and the fraction of EdU-positive cells in the inhibitor group was 20.0%, which was significantly lower than that in the control group (29.4%) (P <0.05) (fig. 3a, b). The results show that the miR-183 mimics promote BRL-3A cell proliferation, and the inhibitor inhibits BRL-3A cell proliferation.

Example 8: target gene prediction of miRNAs and functional confirmation thereof

According to the expression change of the miRNAs in the liver regeneration of rats, miRNAs with meaningful expression change are found. The target genes of the miRNAs are predicted by using an online analysis tool such as TargetScan, MiRanda, PciTar, MiRDB, MiRWalk and the like. Briefly, in miRWalk (www.umm.uni-heidelberg).

Html) on the homepage of de/apps/zmf/mirqwalk/index, clicking the "gene targets" of the "MicroRNA targets" column, inputting the target miRNAs into the SEARCH column, selecting TargetScan, miRanda, PciTar, miRDB, miRWalk from the database options, clicking "SEARCH", and obtaining the target gene. Html website is further searched for KEGG (www.genome.Jp/keg/pathway) to confirm target genes of miRNAs. And (3) comparing the confirmed target gene with a GO database, and finding out a signal pathway and/or cell proliferation and apoptosis activities in which the target gene participates. And continuously inputting the target genes participating in the cell proliferation and apoptosis signal pathways into IPA software and Qiagen databases to obtain the structures, components and signal conduction pathways of the signal pathways.

Example 9: cell cycle assays for modulation of modulators of the invention

To examine the effect of the miR-183 mimetics on the cell cycle of rat normal hepatocytes BRL-3A, cells were seeded in 24-well cell culture plates at 1X 10⁵And (3) adding Lipofectamine 2000(Invitrogen) and a simulant and a control or an inhibitor and a control to transfect the cells when the cells grow to 50-60% confluence, wherein the transfection concentration is as follows: both mock and control concentrations were 100 nM/well, and both inhibitor and control concentrations were 150 nM/well. At 5% CO₂And culturing in an incubator at 37 ℃ for 48 hours, and then collecting cells. Cells were fixed with 70% ethanol overnight at-20 deg.C, washed with PBS and filtered through a 200 mesh screen, followed by PI staining solution (50. mu.g/mLPI, 100. mu.g/mL DNase-free RN)aseA) was stained at room temperature in the dark for 30min and the cell cycle was examined by flow cytometry.

The flow cytometry detection result shows that the cell proliferation rate (namely the S-phase cell ratio) of the miR-183 imic treatment group and the control group thereof is 19.10% and 12.10% respectively (p is less than 0.05), and the cell proliferation rate (S + G2/M%) of the inhibitor treatment group and the control group thereof is 12.30% and 16.20% respectively (p is less than 0.05). The above results show that miR-183 promotes cell cycle progression of BRL-3A and inhibitor inhibits cell cycle progression of BRL-3A, as shown in FIG. 4.

Example 10: design and construction of dual-luciferase reporter gene detection system

Preparing a suppressor (namely SEQ ID NO.2), changing CACTGGA in a partial suppressor sequence SEQ ID NO.2 into TATTAA, changing the sequence by a mutation method or a re-synthesis method, and marking the changed sequence as a suppressor mutation sequence, namely the suppressor mutation sequence only changes CACTGGA in the SEQ ID NO.2 into TATTAA, and other base sequences are consistent with the suppressor SEQ ID NO. 2.

Then, the inhibitor (SEQ ID NO.2) and the inhibitor mutation sequence are respectively cloned on a psi-CHECKM-2 carrier to obtain a corresponding carrier (psi-CH-tnf-wt) containing the miR-183 inhibitor, a carrier (psi-CH-tnf-mut) containing the miR-183 inhibitor mutation sequence and the psi-CHECKM-2 carrier as a reference (psi-CH).

Example 11: mechanism for promoting BRL-3A cell growth and proliferation by miR-183

Taking BRL-3A cells in logarithmic growth phase at 5 × 10⁴The seed/well is inoculated in a 24-well plate, and is co-transformed with miR-183mimic/inhibitor by using psi-CH-tnf-wt, psi-CH-tnf-mu and psi-CH2 respectively. At 48h post-transfection, the protein extracts were analyzed for fluorescence using the dual luciferase reporter assay kit (Promega). The results show that the miR-183mimic can bind to the 3'-UTR of Tnfrfsf 1 alpha and reduce the fluorescence activity (p <0.05), but the miR-183mimic has reduced binding to the 3' -UTR of Tnfrfsf 1 alpha and no reduction in fluorescence activity (p <0.05) is observed after the miR-183 inhibitor (SEQ ID NO.2) is added, as shown in FIG. 5.

The result shows that the miR-183mimic can inhibit the expression of Tnfrsf1 alpha by targeting and combining with the 3' -UTR of Tnfrsf1 alpha, thereby inhibiting the apoptosis of BRL-3A cells and promoting the proliferation of BRL-3A. And the inhibitor of miR-183 can inhibit the combination of miR-183 and 3' -UTR of Tnfrfsf 1 alpha, promote the expression of Tnfrfsf 1 alpha and promote the apoptosis of BRL-3A cells.

In the invention, the principle of promoting cell proliferation in the miR-183mimic is to introduce the mimic into a receptor cell by a gene transfection method, and the mimic is combined with the 3' -UTR of Tnfrsf1 alpha to reduce the expression level of Tnfrsf1 alpha, so that the apoptosis promoting effect of Tnfrsf1 alpha is blocked and the cell proliferation is promoted; the principle of miR-183 inhibitor in vivo action is to design and construct a recombinant vector, wherein the recombinant vector contains an inhibitor nucleotide sequence of miR-183, the recombinant vector is introduced into a receptor cell by a gene transfer method, the inhibitor nucleotide sequence of miR-183 is overexpressed by the vector, and the inhibitor nucleotide sequence is in fixed point combination with miR-183, so that the combination of miR-183 and 3' -UTR of Tnfrfsf 1 alpha is inhibited, the expression level of Tnfrfsf 1 alpha is increased, and the apoptosis promoting effect of Tnfsf 1 alpha is promoted.

The above is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be conceived by those skilled in the art within the technical scope of the present invention disclosed herein are intended to be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

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Claims

1. An application of a miR-183mimic in preparing a medicine for promoting normal liver cell proliferation, wherein the base sequence of the miR-183mimic is as follows: SEQ ID NO. 1.

The application of the miR-183 inhibitor in preparing the medicine for inhibiting the normal liver cell proliferation is that the base sequence of the miR-183 inhibitor is as follows: SEQ ID NO. 2.

3. Use according to claim 2, characterized in that: the inhibitor is capable of binding to the miR-183, and the binding site of the inhibitor to the miR-183 is GTGCCATC.

4. Use according to claim 2, characterized in that: the nucleotide sequences of the inhibitors are SEQ ID NO.2, wherein restriction sites are added to the 5 'and 3' ends of the inhibitors, and the restriction sites are sites which generate sticky ends after enzyme digestion.

5. Use according to claim 2, characterized in that: the medicine also comprises a recombinant expression vector which contains the miR-183 inhibitor.

6. Use according to claim 5, characterized in that: the marker gene in the recombinant expression vector is a dual-luciferase gene.

7. Use according to claim 5, characterized in that: the recombinant expression vector is obtained by connecting the miR-183 inhibitor to a psi-CHECK-2 vector.