CN109394779A

CN109394779A - A kind of miR-183 instrumentality of liver cell and its application adjusted in liver cell drug is treated in preparation

Info

Publication number: CN109394779A
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: 2019-03-01
Anticipated expiration: 2038-10-17
Also published as: CN109394779B

Abstract

The present invention relates to a kind of miR-183 instrumentalities for adjusting liver cell, and the application adjusted in liver cell drug is treated in preparation, and instrumentality includes the instrumentality for inhibiting Tnfrsf1 alpha expression and/or promoting hepatocyte growth correlative protein expression；Adjusting liver cell drug is the drug containing the instrumentality so that the instrumentality can play a role.Instrumentality includes the miR-183 analogies that liver cell can be promoted to be proliferated.Instrumentality further includes the mortifier that can promote liver cell apoptosis.The instrumentality of liver cell can be adjusted the present invention provides one kind and its application adjusted in liver cell hyperproliferation agent is treated in preparation, the applicant has found the instrumentality that can adjust liver cell proliferation or apoptosis by research, that is miR-183 analogies and mortifier, the two is respectively provided with the significant effect for promoting hepatocyte growth and inhibiting hepatocyte growth, therefore can be made into corresponding drug, to realize the adjusting to liver cell.

Description

A kind of miR-183 instrumentality of liver cell and its treat that adjust liver thin in preparation Application in born of the same parents' drug

Technical field

The invention belongs to biomedicine technical fields, and in particular to one kind can adjust the mould of the miR-183 of liver cell Quasi- object, mortifier, recombinant expression carrier of mortifier and application thereof.

Background technique

Liver is the vitals of body, has storage, metabolism, bioconversion, removing toxic substances, hematopoiesis, synthesis BILE PIGMENTS, divides The functions such as secrete, regenerate.Study effect of the genes related with liver regeneration to hepatocyte growth and liver regeneration, to disclose liver regeneration mechanism, Building artificial liver, foundation treatment and prevention hepatopathy method etc. have most important theories meaning and application value.

MicroRNA (miRNA) is that a kind of non-coding for being about 22 nucleotide by the length of endogenous gene is single-stranded Up to the present RNA molecule has been found that there are 28645 miRNA molecules in animals and plants and virus.MiRNA is identified simultaneously The mRNA 3'UTR of target gene transcription is connected, and by the specific recognition area on its specific binding site combination 5'UTR, so Silencing complex is formed with a series of functional proteins afterwards, the degradation of mRNA is induced, to block translation and the albumen of target gene In the expression of post-transcriptional level.Different according to the function of target gene, miRNA may participate in cell cycle, differentiation and apoptosis, increasingly Mostly by the great attention of researcher.

MiR-183 is one of miR-183 family (including miR-183, miR-182 and miR-96), by the study found that Expression of the miR-183 in the tumor tissues such as colorectal cancer, prostate cancer is higher than peritumoral tissues, can be used as tumour and examines in early days Disconnected and prognosis evaluation one of index.It is healthy right that miR-183 is higher than in the expression of cirrhosis, precancerous lesion and liver cancer tissue According to group, and the expression by inhibiting its target gene AKAP12 gene, participate in the occurrence and development process of liver cancer.A variety of biological informations It learns software prediction and shows that miR-183 is adjustable the expression of multiple target genes such as MTA1, Tnfrsf1 α, PDCD4 and AKAP12.Its In, the coded product of Tnfrsf1 α gene is TNF-α inducing hepatocyte apoptosis major receptors.When by various damages, liver is thin Great expression TNFRI on after birth, Tnfrsf1 α are in key position in TNF-α induction acute liver failure.

Summary of the invention

Adjusting liver cell drug is treated the present invention provides a kind of miR-183 instrumentality of liver cell and its in preparation In application, the analogies and mortifier can be used for adjusting Tnfrsf1 α protein expression, and realize adjust hepatocyte growth or The preclinical medicine of apoptosis and clinical medical research, be conducive to for exploitation related drugs provide efficient, big flux screening and Evaluation platform and means will greatly promote application of the miRNA in tumor prevention, diagnosing and treating.

The purpose of the present invention is what is be achieved through the following technical solutions:

A kind of miR-183 instrumentality of liver cell treats the application adjusted in liver cell drug, the tune in preparation Section object includes the instrumentality for inhibiting Tnfrsf1 alpha expression and/or promoting hepatocyte growth correlative protein expression；The adjusting liver Cell drug is the drug containing the instrumentality so that the instrumentality can play promotion and/or inhibit liver cell proliferation Effect.

Further, the instrumentality includes the miR-183 analogies that liver cell can be promoted to be proliferated, the miR- The base sequence of 183 analogies are as follows: SEQ ID NO.1, the SEQ ID NO.1:uauggcacug guagaauuca cu.

Further, the instrumentality further includes the mortifier that can promote liver cell apoptosis, the alkali of the mortifier Basic sequence are as follows: SEQ ID NO.2, the SEQ ID NO.2 are as follows: 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。

Further, the mortifier is the mortifier that can be combined with the miR-183 analogies, the inhibition The binding site of object and the miR-183 analogies is GTGCCATC；If the two is combined by the sequence site, can extremely have The effect of the inhibition miR-183 analogies of effect, to release the effect of the rush liver cell apoptosis of Tnfrsf1 α, therefore real Now promote the purpose of liver cell apoptosis.

Further, restrictive is added in the end 5' and 3' in the base sequence SEQ ID NO.2 of the mortifier Restriction enzyme site, the restriction enzyme site are the restriction enzyme site that cohesive end is generated after digestion；

Further, the restriction enzyme site is respectively Xho I and the corresponding nucleotide sequence of Not I；The Xho I restriction enzyme site is located at the end 5', and I restriction enzyme site of Not is located at the end 3'.

Further, the instrumentality further includes the recombinant expression carrier that can promote liver cell apoptosis, the recombination It include the mortifier in expression vector.

Further, the marker gene in the recombinant expression carrier is Dual-Luciferase.

Further, the recombinant expression carrier is by by the miR- as described in any one of claim 3-5 183 mortifiers are connected to recombinant expression carrier obtained in psi-CHECK-2 (i.e. psi-CHECKTM-2) carrier.

A kind of instrumentality that can adjust liver cell, the instrumentality are instrumentality described above.

A kind of instrumentality that can adjust liver cell is promoting, is inhibiting application in liver cell proliferation, the adjusting Object be instrumentality described in any of the above embodiments, the instrumentality include can promote liver cell be proliferated miR-183 analogies, It is able to suppress the mortifier of liver cell proliferation or is able to suppress the recombinant expression carrier of liver cell proliferation, referring specifically to above It limits.

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

Adjusting liver cell drug is treated the present invention provides a kind of miR-183 instrumentality of liver cell and its in preparation In application, the applicant by study pleasantly surprised having found can adjust liver cell proliferation or apoptosis instrumentality, i.e., MiR-183 analogies and mortifier, the two are respectively provided with the significant effect for promoting hepatocyte growth and inhibiting hepatocyte growth, Therefore can be made into corresponding drug, to realize the adjusting to liver cell.

The miR-183 analogies being previously mentioned can be used for reducing the expression of Tnfrsf1 α albumen, to promote liver cell Proliferation, therefore, miR-183 analogies can be applicable to promote hepatocyte growth preclinical medicine and clinical medical research, Drug can be made further to realize the purpose for promoting hepatocyte growth.Mortifier and its recombinant expression carrier can be used for inhibiting MiR-183 increases the expression of Tnfrsf1 α albumen, to promote the apoptosis of liver cell, therefore, which be can be applicable to The preclinical medicine and clinical medical research for promoting hepatocellular apoptosis, can also further be made drug to realize to liver cell It adjusts.The two is conducive to the screening that efficient, big flux is provided for exploitation related drugs and evaluation platform and means, will promote significantly Into application of the miRNA in tumor prevention, diagnosing and treating.

Detailed description of the invention

Fig. 1 is the high-throughput testing result of miR-183 compared with qRT-PCR testing result；

Fig. 2 is that the influence of the mimic and inhibitor of miR-183 to BRL-3A cell-proliferation activity is detected with mtt assay；

Fig. 3 a is that EdU method detects the influence of the mimic and inhibitor of miR-183 to BRL-3A cell-proliferation activity；

Fig. 3 b is the mimic and inhibitor of miR-183 to BRL-3A cell-proliferation activity statistic analysis result；

Fig. 4 is the influence of the mimic and inhibitor of miR-183 to the BRL-3A cell cycle；

Fig. 5 is the combination of the 3'-UTR of Dual-Luciferase method validation miR-183 and Tnfrsf1 α.

Specific embodiment

The invention will be further described with reference to the accompanying drawings and examples, following embodiment be it is descriptive, be not Limited, this does not limit the scope of protection of the present invention.

Embodiment 1: the preparation of Liver Regeneration of Rat model and materials

Experimental rat is adult healthy male Sprague-Dawley rat, 230 ± 20g of weight, by He'nan Normal University Experimental Animal Center provides.Raising temperature is 21 ± 2 DEG C, and relative humidity is 60 ± 10%, light application time 12h/d (8:00-20: 00), free water, ingest.114 above-mentioned rats are taken, are randomly divided into 19 groups, every group 6.Wherein, 1 group of normal control (0h), It is 9 groups each that 2/3 hepatectomy (partial hepatectomy, PH) compares (sham operation, SO) with sham-operation.PH group is pressed Higgins and Anderson method carries out, and SO group is not in addition to cutting off lobe of the liver, other same PH.2 after operation, 6,12,24,30,36, 72, it when 120 and 168h, takes right lobe of liver to be placed in tissue storage reagent (such as RNALater), is saved backup in -20 DEG C.

The high-flux sequence of embodiment 2:miRNAs

It takes the appropriate above-mentioned hepatic tissue for being stored in -20 DEG C to be placed in the mortar equipped with liquid nitrogen to grind, by kit (mirVana miRNAIsolation Kit, Ambion, USA) operating guidance extracts and purifying miRNAs.Use Ago-Gel Electrophoresis (180V, 0.5h) detect total serum IgE quality, 28S rRNA:18S rRNA is about 2:1, and when measure OD respectively₂₆₀With OD₂₈₀, in OD₂₆₀/OD₂₈₀>=2.0, it is considered as RNA qualification.The qualitative and quantitative analysis of miRNAs is public by the bold and unconstrained biotechnology of Shanghai uncle Department carries out, and sequencing approach is single-ended Solexa microRNA-Seq sequencing, reads long 36nt.The miRNAs sequence that sequencing is obtained It is compared with the library miRNAs, determines the type and abundance of miRNAs.

Embodiment 3: Liver Regeneration of Rat correlation miRNAs

With single-ended Solexa microRNA-Seq high-flux sequence method, from the 0 of experimental group (PH) and sham-operation group (SO), 2,425 miRNAs are detected in the Regenerating Liver of Rat at 10 time points such as 6,12,24,30,36,72,120 and 168h, are passed through Ratio value analysis shows, in 126 miRNAs of the signal value greater than 20,39 have occurred significant expression variation.Wherein, 31 ratio values >=2 times of control are considered as significant expression up-regulation.4 ratio values≤2 times of control, are considered as significant table Up to downward.4 are lowered at the time point up-regulation having, some time points, are considered as up/down tune.T- inspection shows above-mentioned 39 hairs In the miRNAs for having given birth to significant expression variation, 23 miRNAs (table 1) related to liver regeneration.

The miRNAs of significant transcription variation occurs in 1 Liver Regeneration of Rat of table

Note:Indicate value >=2 ratio；Indicate value≤0.5 ratio；It indicates P < 0.05, speculates For Liver Regeneration of Rat correlation microRNAs.

Embodiment 4: real-time fluorescence quantitative PCR (qRT-PCR) detection

Design specificity Stem-Loop reverse transcription (RT) primer (SEQ ID NO.3), qRT-PCR upstream and downstream primer sequence With U6 internal control primer sequence, qRT-PCR primer includes that miR-183 upstream primer (SEQ ID NO. 4) and the downstream miR-183 are drawn Object (SEQ ID NO.5), U6 internal control primer sequence include U6 upstream primer (SEQ ID NO.6) and U6 downstream primer (SEQ ID NO.7), and species are carried out with miRBase to compare, determine the specificity of primer, each primer sequence is as shown in table 2.

Reagent (Trizol) operational manual (Invitrogen, USA), which is extracted, by RNA carries out cell total rna extracting, point Light photometric determination OD₂₆₀And OD₂₈₀, in OD₂₆₀/OD₂₈₀>=2.0, and Denaturing Agarose Gel electrophoresis (70V, 20min) detects 28S rRNA:18S rRNA is considered as RNA qualification when being about 2:1.Using 2 μ g RNA as template, by AMV reverse transcription reagent box The operating instruction of (Promega, USA) carries out reverse transcription, obtains the first chain cDNA.Then, 1 μ l cDNA is taken, 10 μ l fluorescence are added Dye mixture (I Mix of SyBr Green), 0.4 μ l primer, 8.6 μ l remove the pure water of nuclease.After mixing, it is put into fluorescent quantitation Amplification gene in PCR instrument (Rotor-Gene 3000) (Corbett Robotics, Australia), detects amplified production Fluorescence signal value, and be the relative expression quantity (ratio value) that internal reference calculates gene with β-actin (NM_031144).qRT-PCR Condition it is equal are as follows: 95 DEG C of 2min, 95 DEG C of 15sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 40 circulation.Each sample repeats to detect Three times, the data obtained is with 2^-ΔΔCtMethod makees relative quantification processing.

2 reverse transcription primer of table and PCR primer sequence

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

Variation is expressed in Regenerating Liver of Rat with qRT-PCR verifying miR-183, verification result is as shown in Figure 1, miR-183 Expression trend and high-flux sequence result coincide substantially.

Embodiment 5: rat hepatocytes culture

It tests rat normal liver cell BRL-3A used and is purchased from Shanghai Inst. of Life Science, CAS cellular resources Center, culture medium are the DMEM high glucose medium containing 10% fetal calf serum (FBS), and cell culture is in 37 DEG C, saturated humidity, 5% CO₂Incubator in.Logarithmic growth phase cell is passed on, and 5 × 10⁴A cell/bottle.

The detection of embodiment 6:MTT method is by instrumentality of the present invention treated hepatocyte growth situation

The BRL-3A cell of logarithmic growth phase, 0.25% pancreatin (Invitrogen, USA) digestion by the hole 1ml/, contain 5 ×10⁴The cell suspension inoculation of a/ml cell is in 24 porocyte culture plates, after cultivating 12h, by lipofectamine The operational manual of (Lipofectamine RNAiMAX, Invitrogen, USA) carries out cell transfecting.In short, existing respectively MiR-183 mimic (miR-183 analogies) (100nM), mimic NC (analogies are added in 25 μ l OPTI-MEM culture mediums Negative control) (150nM), inhibitor (mortifier) (100nM), inhibitorNC (mortifier negative control) (150nM) With 1.5 μ l transfection reagents, it is stored at room temperature 5min.Above-mentioned solution is mixed gently, transfection composite is formed, is stored at room temperature 20min is added in the cell of the culture medium of OPTI-MEM containing 0.45ml (Gibco, USA), and 37 DEG C of incubation 4h change normal culture Base.Experiment is repeated 3 times, and 3 multiple holes are arranged in each experimental group.

10 μ l/ hole MTT (Geneview, USA) are added in 96 orifice plates containing cell and culture medium, reach its ultimate density 0.5g/L is protected from light culture 4h in 37 DEG C, after thoroughly discarding culture solution, 100 μ l dimethyl sulfoxides of every hole addition (DMSO, Geneview, USA), 10min is gently shaken, first a ceremonial jade-ladle, used in libation crystal is sufficiently dissolved.Finally, with microplate reader (Biotek, USA) in 490nm The light absorption value in each hole is detected at wavelength.Experiment is repeated 3 times, and 3 multiple holes are arranged in each experimental group.

Testing result is as shown in Fig. 2, miR-183mimic processing group, cell-proliferation activity are higher than mimic NC group, explanation The proliferation of miR-183mimic promotion BRL-3A cell；Inhibitor processing group, cell-proliferation activity are lower than inhibitorNC Group illustrates that inhibitor inhibits BRL-3A cell Proliferation (P < 0.05).

The detection of embodiment 7:EdU labelling method is by instrumentality of the present invention treated hepatocyte growth situation

The BRL-3A cell of logarithmic growth phase, by 5 × 10⁴The cell density in/hole is inoculated in 24 holes of preset round slide In plate, after cultivating 12h, the BRL-3A cell of mimic (100nM) and inhibitor (150nM) transfection in vitro culture, after transfection 48h takes out cell climbing sheet, and EdU (sharp rich, Guangzhou), which is added, in 2h before drawing materials makes its final concentration of 50 μM, and 4% paraformaldehyde is fixed 30min.Decoloration is incubated for 5min in glycine solution (2g/L).Then, it decolourizes to be incubated for then at 0.5%TritonX-100 10min.Then, it is incubated for 30min in 1 × Apollo staining reaction liquid (sharp rich, Guangzhou), then at 0.5%TritonX-100 It is incubated for 10-30min, and incubation at room temperature 30min marks nucleus in 1 × Hoechst, 33342 reaction solution (sharp rich, Guangzhou), One step of above-mentioned every progress, is washed 3 times with PBS, each 5min.Finally, randomly selected under fluorescence microscope 5 it is nonoverlapping The visual field (20 ×), is observed and is taken pictures, and with 6.0 software of Image-Pro Plus under EdU positive cell and corresponding visual field Nucleus counted respectively.And group difference is analyzed with the one-way analysis of variance method of 13.0 statistics software of SPSS.

Testing result shows that mimic group EdU positive cell ratio is 37.5%, hence it is evident that is higher than its control group (30.5%) (P < 0.05), the EdU positive cell ratio of inhibitor group are 20.0%, hence it is evident that are lower than its control group (29.4%) (P < 0.05) (Fig. 3 a, b).The above results show that miR-183 analogies promote BRL-3A cell Proliferation, and mortifier inhibits BRL-3A Cell Proliferation.

The microRNA target prediction and its function of embodiment 8:miRNAs confirms

According to expression variation of the miRNAs in Liver Regeneration of Rat, the miRNAs that significant expression variation occurs is found out.With The on-line analyses tool such as TargetScan, miRanda, PciTar, miRDB, miRWalk predicts the target gene of above-mentioned miRNAs. In brief, in miRWalk (www.umm.uni-heidelberg.

De/apps/zmf/mirqwalk/index.html " the gene on the column " MicroRNA targets ") is clicked on homepage Targets ", by purpose miRNAs input search column, in Database Options select TargetScan, miRanda, PciTar, MiRDB, miRWalk are clicked " SEARCH ", obtain target gene.Further search for KEGG (www.genome.Jp/keg/ Pathway.html) website confirms the target gene of miRNAs.The target gene that will confirm that is compared with GO database, finds out target gene Signal path and/or cell Proliferation and the apoptosis activity of participation.Continue will to participate in the target base of cell Proliferation and apoptotic signal access Because of databases such as softwares and Qiagen such as input IPA, structure, ingredient and the signal transduction path of signal path are obtained.

Embodiment 9: the cell cycle detects the adjusting situation of instrumentality of the present invention

Influence for detection miR-183 analogies to the cell cycle of rat normal liver cell BRL-3A, by cell inoculation In 24 porocyte culture plates, 1 × 10⁵A cells/well is added when cell grows to 50~60% convergence degree Lipofectamine 2000 (Invitrogen) and analogies and control or mortifier and control transfection cell, transfection concentrations Are as follows: analogies and its control concentration are the hole 100nM/, and mortifier and its control concentration are the hole 150nM/.In 5%CO₂、37 Cell is collected after cultivating 48h in DEG C incubator.With 70% ethyl alcohol in -20 DEG C of fixed cell pellet overnights, washed and 200 meshes with PBS After net filtration cell, then with PI dye liquor (50 μ g/mLPI, 100 μ g/mL DNase-free RNaseA) room temperature it is protected from light dyeing 30min, with the Flow cytometry cell cycle.

Flow cytometry the results show that miR-183mimic processing group cell proliferation rate (the i.e. S with its control group Phase cell proportion) it is respectively 19.10% and 12.10% (p < 0.05), the cell with its control group of inhibitor processing group Proliferation rate (S+G2/M%) is respectively 12.30% and 16.20% (p < 0.05).The above result shows that as shown in figure 4, miR- 183 promote the cell cycle progression of BRL-3A, and inhibitor inhibits the cell cycle progression of BRL-3A.

Embodiment 10: the design and building of luciferase reporter gene detection system

Prepare mortifier (i.e. SEQ ID NO.2), the CACTGGA in the mortifier sequence SEQ ID NO.2 of part is become TATTAA is changed using the method or the method that recombines of mutation, and the sequence after change is denoted as mortifier mutant nucleotide sequence, I.e. the CACTGGA in SEQ ID NO.2 is only become TATTAA by the mortifier mutant nucleotide sequence, other base sequences with suppression Object SEQ ID NO.2 processed is consistent.

Then mortifier (SEQ ID NO.2), mortifier mutant nucleotide sequence are cloned into psi-CHECKTM-2 carrier respectively On, obtain the carrier (psi-CH-tnf-wt) of the mortifier containing miR-183 accordingly, mortifier mutant nucleotide sequence containing miR-183 Carrier (psi-CH-tnf-mut), psi-CHECKTM-2 carrier is as control (psi-CH).

The promotion BRL-3A growth and proliferation of cell mechanism of embodiment 11:miR-183

The BRL-3A cell of logarithmic growth phase, by 5 × 10⁴/ hole is seeded in 24 orifice plates, with psi-CH-tnf-wt, Psi-CH-tnf-mu, psi-CH2 respectively with miR-183mimic/inhibitor corotation.48h after transfection uses double fluorescence The fluorescent value of plain enzyme reporter assay kit (Promega) analysis protein extract.The result shows that miR-183 is simulated Object can be in conjunction with the 3'-UTR of Tnfrsf1 α, and reduces fluorescence activity (p < 0.05), but adds miR-183 mortifier (SEQ ID NO.2 after), miR-183 is reduced in conjunction with the 3'-UTR of Tnfrsf1 α, also has no that fluorescence activity reduces (p < 0.05), such as Fig. 5 It is shown.

This is the result shows that miR-183 analogies can check Tnfrsf1 α by targeting the 3'-UTR in conjunction with Tnfrsf1 α Expression, to inhibit BRL-3A Apoptosis, promotes the proliferation of BRL-3A.And the inhibitor of miR-183 can inhibit MiR-183 promotes Tnfrsf1 alpha expression in conjunction with the 3'-UTR of Tnfrsf1 α, promotes BRL-3A Apoptosis.

In the present invention, the principle for promoting cell Proliferation in the analogies body of miR-183 is the method that is transfected with gene by mould Quasi- object imports recipient cell, which leads to the reduction of Tnfrsf1 alpha expression level in conjunction with the 3'-UTR of Tnfrsf1 α, thus The apoptosis-promoting effect of Tnfrsf1 α is checked, cell Proliferation is promoted；The principle to play a role in the mortifier body of miR-183 is design And recombinant vector is constructed, the interior mortifier nucleotide sequence containing miR-183 is led recombinant vector with the method for gene transfer Enter recipient cell, the mortifier nucleotide sequence of miR-183, this mortifier nucleotide sequence and miR- are overexpressed by carrier 183 fixed points combine, to inhibit miR-183 in conjunction with the 3'-UTR of Tnfrsf1 α, Tnfrsf1 alpha expression level increases, to promote Into the rush cells apoptosis of Tnfrsf1 α.

More than, it is merely preferred embodiments of the present invention, but the protection scope invented is not limited thereto, it is any ripe Those skilled in the art are known in the technical scope revealed of the present invention, it is contemplated that change or replacement, should all cover Within protection scope of the present invention.Therefore, the scope of protection of the invention shall be subject to the scope of protection specified in the patent claim.

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Claims

1. the miR-183 instrumentality that one kind can adjust liver cell treats the application adjusted in liver cell drug in preparation, It is characterized in that, the instrumentality includes the tune for inhibiting Tnfrsf1 alpha expression and/or promoting hepatocyte growth correlative protein expression Save object；The adjusting liver cell drug be the drug containing the instrumentality so that the instrumentality can play promotion and/ Or inhibit the effect of liver cell proliferation.

2. application according to claim 1, it is characterised in that: the instrumentality includes that liver cell can be promoted to be proliferated MiR-183 analogies, the base sequence of the miR-183 analogies are as follows: SEQ ID NO.1.

3. application according to claim 1 or 2, it is characterised in that: the instrumentality further includes that can promote liver cell The mortifier of apoptosis, the base sequence of the mortifier are as follows: SEQ ID NO.2.

4. application according to claim 3, it is characterised in that: the mortifier is that can be tied with the miR-183 The binding site of the mortifier of conjunction, the mortifier and the miR-183 are GTGCCATC.

5. application according to claim 3, it is characterised in that: in the base sequence SEQ ID NO.2 of the mortifier The end 5' and 3' is added with restriction enzyme site, which is the limitation that cohesive end is generated after digestion Property restriction enzyme site；

Preferably, the restriction enzyme site is respectively Xho I and the corresponding nucleotide sequence of Not I；I enzyme of Xho Enzyme site is located at the end 5', and I restriction enzyme site of Not is located at the end 3'.

6. application according to claim 3, it is characterised in that: the instrumentality further includes that can promote liver cell apoptosis Recombinant expression carrier, include the mortifier in the recombinant expression carrier.

7. application according to claim 6, it is characterised in that: the marker gene in the recombinant expression carrier is double fluorescence Plain enzyme.

8. application according to claim 7, it is characterised in that: the recombinant expression carrier is by will be such as claim 3- MiR-183 mortifier described in any one of 5 is connected to recombinant expression carrier obtained in psi-CHECK-2 carrier.

9. the instrumentality that one kind can adjust liver cell, which is characterized in that the instrumentality is any one in claim 1-8 Instrumentality described in.

10. one kind can adjust application of the instrumentality of liver cell in promotion, inhibition liver cell proliferation, the instrumentality For instrumentality of any of claims 1-8, the instrumentality includes the miR-183 that liver cell can be promoted to be proliferated Analogies, the mortifier for being able to suppress liver cell proliferation or the recombinant expression carrier for being able to suppress liver cell proliferation.