Screening method of target gene for regulating astrocyte proliferation by miR-31 and application thereof

Abstract

The invention discloses a screening method of a target gene for regulating astrocyte proliferation by miR-31 and application of the screening method, and belongs to the technical field of biomedicines.The screening method comprises the following steps that S1, astrocytes are separated, specifically, the astrocytes are separated and cultured, astrocyte markers are identified through an immunofluorescence method, and the cell purity is detected; s2, establishing a miR-31 overexpression model: breeding and identifying a miR-31 overexpression positive mouse through genetic engineering, and constructing a miR-31 overexpression astrocyte model at the cellular level through a transfection technology; s3, verifying transfection efficiency through real-time fluorescent quantitative PCR (polymerase chain reaction): verifying the transfection efficiency of miR-31 and the influence of miR-31 on cell proliferation by adopting RT-qPCR and CCK-8 experiments; and S4, carrying out molecular level verification and functional verification. According to the invention, LATS2 is successfully positioned as a key target gene of miR-31, and the problems of unclear regulation and control mechanism of astrocyte proliferation and limitation of miRNA targeting prediction in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for screening a target gene for regulating astrocyte proliferation by miR-31 and an application thereof. Background Art

[0002] Spinal cord injury is a serious traumatic disease of the central nervous system (CNS). Trauma, tumors or inflammation and other factors lead to the destruction of the integrity and continuity of the spinal cord, resulting in motor, sensory and autonomic dysfunction below the level of injury and even the failure of other organs in the body. Spinal cord injury usually occurs in two stages. The first stage is the acute phase injury caused directly by trauma, followed by the subacute phase, in which various factors such as excitotoxic injury, hemorrhage, ischemia and inflammatory changes and subsequent cell necrosis or apoptosis, long-term Wallerian degeneration and scar formation lead to secondary damage to the spinal cord tissue, severely limiting injury recovery and even aggravating functional loss. According to the World Health Organization, 250,000 to 500,000 people are affected by spinal cord injury every year, and currently more than 1 million patients worldwide are paralyzed due to spinal cord injury. In recent years, the incidence of spinal cord injury has increased year by year, reaching approximately 40-80 cases per million people, and existing treatments cannot effectively restore motor function after spinal cord injury. In addition to high treatment costs and poor prognosis, it can also cause serious physical and psychological harm to patients, bringing a huge burden to families and society.

[0003] Treatment after injury has always been a difficult problem that has troubled people and a hot topic of research. At present, the clinical treatment of spinal cord injury is limited to surgical decompression, hormone shock therapy, and neuroprotective therapy, but its clinical efficacy is very limited. Patients mainly maintain their remaining neurological functions through late rehabilitation training. This is mainly due to the poor plasticity of the central nervous system and the limited inherent regeneration capacity of neurons. The regeneration and repair after spinal cord injury is extremely weak. There is currently no effective clinical treatment measure that can completely restore the lost neurological function after spinal cord injury. Regeneration and repair has become a world problem that needs to be solved urgently in today's medical community. Finding methods that are conducive to neuronal regeneration has become a research focus in the field of spinal cord injury treatment.

[0004] Therefore, there is an urgent need for a method for screening target genes that regulate astrocyte proliferation by miR-31 and its application, aiming to break through the current treatment bottleneck, promote neurological function recovery, and open up new avenues for the treatment of spinal cord injury. Summary of the invention

[0005] To this end, the present invention provides a method for screening target genes for miR-31 regulating astrocyte proliferation and its application, so as to solve the problems in the prior art that the astrocyte proliferation regulation mechanism is unclear and miRNA targeting prediction has certain limitations.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] According to the first aspect of the present invention, a target gene for miR-31 regulating astrocyte proliferation is provided, and the key downstream target gene for miR-31 regulating astrocyte proliferation is LATS2. The sequence of miR-31 is shown in SEQ ID NO.1, and the sequence of LATS2 is shown in SEQ ID NO.2.

[0008] According to a second aspect of the present invention, a method for screening target genes for miR-31 regulating astrocyte proliferation is provided, comprising the following steps:

[0009] S1. Isolation of astrocytes: Isolate and culture astrocytes, identify astrocyte markers and detect cell purity by immunofluorescence;

[0010] S2. Establish a miR-31 overexpression model: Genetically engineer and identify miR-31 overexpression positive mice, and construct a miR-31 overexpression astrocyte model at the cellular level by transfection technology;

[0011] S3. Real-time fluorescence quantitative PCR to verify transfection efficiency: RT-qPCR and CCK-8 experiments were used to verify the transfection efficiency of miR-31 and its effect on cell proliferation;

[0012] S4. Molecular level verification and functional verification.

[0013] Furthermore, the specific marker of astrocytes is GFAP, and the GFAP is combined with DAPI to label astrocytes to identify and confirm the purity of astrocytes.

[0014] 4. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 2, wherein the separation, culture and purification of astrocytes in S1 comprises the following steps:

[0015] a. Extraction and purification of primary astrocytes: Spinal cord tissue was isolated from 2-3 day old FVB mice, and high-purity primary astrocytes were obtained through digestion, purification and culture;

[0016] b. Passaging: After confirming that the cells are growing well and the confluence reaches 80% under a microscope, wash them twice with PBS, add trypsin to digest for 3-5 minutes, terminate the digestion with complete culture medium, collect the cells by centrifugation, resuspend them, and inoculate them into a new culture flask;

[0017] c. Cryopreservation and thawing:

[0018] After washing the cells with a confluence of 90% twice with PBS, add 2 mL of trypsin to digest for 3 minutes. After the cells become round under the microscope, stop digestion with complete medium, collect the cells by centrifugation and discard the supernatant, freeze them at -80°C with the cryopreservation solution prepared in advance, and inoculate them into culture flasks after rapid thawing and resuspending the cells with complete medium when needed;

[0019] After taking out the cells from the -80℃ freezer or liquid nitrogen, immediately place them in a 37℃ water bath to thaw for 2 minutes, then transfer the cell suspension to a centrifuge tube containing complete culture medium and mix well. After centrifugation, discard the supernatant, resuspend the cells in complete culture medium and inoculate them into the culture flask.

[0020] Furthermore, the immunofluorescence method for identifying cell purity in S1 comprises the following steps:

[0021] A. Preliminary preparation: Soak the coverslip in 75% alcohol overnight, dry it with an alcohol lamp and place it in a six-well plate, prepare a 0.1 mg / mL poly-lysine solution, drop it on the coverslip, place it in an incubator for 4 hours, then aspirate it, wash it 3 times with PBS, place it in an incubator to dry overnight, and prepare BSA;

[0022] B. Cell culture and fixation: The day before, resuspend the cells on coverslips at 3 × 10 cells per well. 5 The cells were placed in an incubator overnight, and the six-well plate was removed from the incubator, the culture medium was discarded, and the plate was washed twice with PBS, fixed with 4% paraformaldehyde for 20-40 min, and washed 2-3 times with PBS;

[0023] C. Immunofluorescence staining:

[0024] Add 200 μL TritonX-100 to each well, permeabilize for 20 min at room temperature, and wash 2-3 times with PBS, each time for 2-5 min;

[0025] Add 1 mL BSA to block for 1 h, and wash 2-3 times with PBS;

[0026] Add primary antibody, incubate at 4°C overnight, let stand at room temperature for 1 hour, wash with PBS 2-3 times, 8-12 minutes each time; add secondary antibody, incubate at room temperature in the dark for 1.5-2.5 hours, wash with PBS 2-3 times, 10 minutes each time;

[0027] Add DAPI and incubate for 10 min, wash 2-3 times with PBS, 55 min each time, and seal the slides;

[0028] D. Observation and calculation: Observe and take pictures under a fluorescence microscope. The cell purity is the ratio of the number of cells stained with GFAP to the number of cells stained with DAPI.

[0029] Furthermore, the construction of the miR-31 overexpressed astrocyte model in S2 comprises the following steps:

[0030] (1) The prepared astrocytes were seeded in a six-well plate and then incubated in a culture medium containing 10% FBS and 1% double antibody for 8-16 h in an incubator;

[0031] (2) diluting the transfection reagent, pipetting 3-5 times to mix, diluting the siRNA, pipetting 3-5 times to mix and letting stand for 3-8 min, mixing the two liquids, pipetting 3-5 times to mix and letting stand at room temperature for 15-25 min to obtain a mixed reagent;

[0032] (3) Remove the cells from the incubator, wash the cells 2-3 times with PBS, and then remove the residual PBS. Add 1500 μL of culture medium containing 12% FBS without dual antibodies to each well, drop the mixed reagent obtained in (2) into the well, shake the plate gently to mix, and replace the complete culture medium containing dual antibodies after 3-6 hours in the incubator. Perform subsequent operations after 12-24 hours.

[0033] Furthermore, in step (2), every 6 μL of Lipofectamine 2000 transfection reagent is diluted with 250 μL of Opti-Mem, and every 5 μL of siRNA is diluted with 250 μL of Opti-Mem.

[0034] Furthermore, the molecular level verification in S4 verifies the miR-31 regulatory mechanism through gene expression analysis, sequencing, and luciferase reporter gene assay, and the functional verification evaluates the functional effect of miR-31 through cell proliferation experiment and protein expression analysis.

[0035] According to a third aspect of the present invention, there is provided a use of miR-31 in the preparation of a medicine or composition for use in a spinal cord injury repair process.

[0036] According to a fourth aspect of the present invention, there is provided a use of miR-31 in the preparation of a medicament or composition for treating a neurodegenerative disease caused by spinal cord injury.

[0037] The present invention has the following advantages:

[0038] 1. The present invention has identified LATS2 as a key downstream target gene for miR-31 to regulate astrocyte proliferation through systematic experimental verification. This discovery not only reveals a new mechanism for motor function recovery after spinal cord injury, but also provides a new target for the development of therapeutic strategies for spinal cord injury and other neurodegenerative diseases involving abnormal astrocyte proliferation. By targeting LATS2, astrocyte proliferation can be regulated, thereby improving the repair process after spinal cord injury.

[0039] 2. The present invention uses RNA-seq technology combined with bioinformatics prediction to successfully screen and verify differentially expressed genes (DEGs) related to miR-31 regulation of astrocyte proliferation. This method provides an efficient way to quickly identify miRNA functions and their regulatory networks, has high technical innovation and application potential, and promotes the development of related research.

[0040] 3. This invention uses a series of experimental techniques to fully verify the interaction between miR-31 and LATS2 and its effect on cell proliferation from the molecular to the cellular level. It ensures the reliability and scientificity of the miR-31 regulatory mechanism through a variety of technical means such as RT-qPCR, CCK-8 experiments, and immunofluorescence. This method not only verifies the effect of miR-31 on astrocyte proliferation, but also provides a solid foundation for future research. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0042] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0043] Figure 1 The volcano map of DEGs provided by the present invention;

[0044] In the figure: A. MiR-31 mimics group and control group; B. MiR-31 inhibitor group and control group;

[0045] Pink: up-regulated DEGs; Green: down-regulated DEGs;

[0046] Horizontal axis: expression difference fold between two samples; vertical axis: significance level of difference between two samples;

[0047] Figure 2 Venn diagram of DEGs provided by the present invention;

[0048] In the figure: Color circles: DEGs downregulated in the MiR-31mimics group; Purple circles: DEGs upregulated in the MiR-31inhibitor group;

[0049] Intersection of two circles: 297 potential target genes;

[0050] Figure 3 GO functional classification annotation diagram of DEGs provided by the present invention;

[0051] In the figure: horizontal axis: gene ratio, the larger the value, the higher the enrichment of differential metabolites / proteins / genes in the pathway;

[0052] Vertical axis: enriched GO or pathway name;

[0053] Figure 4 KEGG classification diagram of DEGs provided by the present invention;

[0054] In the figure: horizontal axis: the ratio of genes located under the pathway set. The larger the value, the higher the degree of gene enrichment in the pathway;

[0055] Vertical axis: pathway sets with higher enrichment;

[0056] Figure 5 Screening and detection of potential target genes of MiR-31 provided by the present invention;

[0057] In the figure:

[0058] A. MiR-31-5p interacts with LATS2 3'UTR sequence;

[0059] B. RT-qPCR verified the relative expression of LATS2 after astrocytes were transfected with different concentrations of miR-31mimics, compared with the NC group ## P<0.01, ### P < 0.001;

[0060] C. RT-qPCR verification of the relative expression of LATS2 in astrocytes after transfection of different concentrations of miR-31inhibitor compared with the NC group ## P<0.01, ### P < 0.001;

[0061] Figure 6 The sequencing result of the recombinant plasmid provided by the present invention;

[0062] In the figure: the sequences of LATS2 WT-miR-31-5p and LATS2 MUT-miR-31-5p plasmids, with the binding sites in the green boxes;

[0063] Figure 7The dual luciferase reporter gene detection analysis of MiR-31-5p and LATS2 provided by the present invention;

[0064] Figure 8 The LATS2 provided by the present invention is involved in astrocyte proliferation;

[0065] In the figure: A, B: RTq-PCR verification of LATS2 expression after transfection, ### P < 0.001;

[0066] C: Flow cytometry was used to measure the EdU ratio of each group to detect cell proliferation. *** P<0.001, compared with NC group # P < 0.05;

[0067] Fig. 9 The relative expression of the LATS2 protein provided by the present invention;

[0068] Figure: A. Bar graph of protein expression; B. Relative expression of LATS2 protein compared with NC group # P < 0.05;

[0069] Fig.10 The role of MiR-31 provided by the present invention in the repair of spinal cord injury;

[0070] In the figure: WT is wild-type FVB mouse; miR-31TG is overexpression positive mouse. DETAILED DESCRIPTION

[0071] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] The present invention provides a target gene for regulating astrocyte proliferation by miR-31, wherein the key downstream target gene for regulating astrocyte proliferation by miR-31 is LATS2, and the sequence of miR-31 is shown in SEQ ID NO.1, specifically 5′-AGGCAAGATGCTGGCATAGCTG-3′;

[0073] The sequence of LATS2 is shown in SEQ ID NO. 2, specifically 5′-GUAUAAAAUACCGUAUCUUGCCA-3′.

[0074] The present invention provides a method for screening target genes for miR-31 regulating astrocyte proliferation, comprising the following steps:

[0075] S1. Isolation of astrocytes: Isolate and culture astrocytes, identify astrocyte markers and detect cell purity by immunofluorescence;

[0076] S2. Establish a miR-31 overexpression model: Genetically engineer and identify miR-31 overexpression positive mice, and construct a miR-31 overexpression astrocyte model at the cellular level by transfection technology;

[0077] S3. Real-time fluorescence quantitative PCR to verify transfection efficiency: RT-qPCR and CCK-8 experiments were used to verify the transfection efficiency of miR-31 and its effect on cell proliferation;

[0078] S4. Molecular level verification and functional verification.

[0079] Specifically, the specific marker of astrocytes is GFAP, and GFAP is combined with DAPI to label astrocytes to identify and confirm the purity of astrocytes.

[0080] Specifically, the isolation, culture and purification of astrocytes in S1 include the following steps:

[0081] a. Extraction and purification of primary astrocytes: Spinal cord tissue was isolated from 2-3 day old FVB mice, and high-purity primary astrocytes were obtained through digestion, purification and culture;

[0082] b. Passaging: After confirming that the cells are growing well and the confluence reaches 80% under a microscope, wash them twice with PBS, add trypsin to digest for 3-5 minutes, terminate the digestion with complete culture medium, collect the cells by centrifugation, resuspend them, and inoculate them into a new culture flask;

[0083] c. Cryopreservation and thawing:

[0084] After washing the cells with a confluence of 90% twice with PBS, add 2 mL of trypsin to digest for 3 minutes. After the cells become round under the microscope, stop digestion with complete medium, collect the cells by centrifugation and discard the supernatant, freeze them at -80°C with the cryopreservation solution prepared in advance, and inoculate them into culture flasks after rapid thawing and resuspending the cells with complete medium when needed;

[0085] After taking out the cells from the -80℃ freezer or liquid nitrogen, immediately place them in a 37℃ water bath to thaw for 2 minutes, then transfer the cell suspension to a centrifuge tube containing complete culture medium and mix well. After centrifugation, discard the supernatant, resuspend the cells in complete culture medium and inoculate them into the culture flask.

[0086] Specifically, the immunofluorescence method for identifying cell purity in S1 includes the following steps:

[0087] A. Preliminary preparation: Soak the coverslip in 75% alcohol overnight, dry it with an alcohol lamp and place it in a six-well plate, prepare a 0.1 mg / mL poly-lysine solution, drop it on the coverslip, place it in an incubator for 4 hours, then aspirate it, wash it 3 times with PBS, place it in an incubator to dry overnight, and prepare BSA;

[0088] B. Cell culture and fixation: The day before, resuspend the cells on coverslips at 3 × 10 cells per well. 5 The cells were placed in an incubator overnight, and the six-well plate was removed from the incubator, the culture medium was discarded, and the plate was washed twice with PBS, fixed with 4% paraformaldehyde for 20-40 min, and washed 2-3 times with PBS;

[0089] C. Immunofluorescence staining:

[0090] Add 200 μL TritonX-100 to each well, permeabilize for 20 min at room temperature, and wash 2-3 times with PBS, each time for 2-5 min;

[0091] Add 1 mL BSA to block for 1 h, and wash 2-3 times with PBS;

[0092] Add primary antibody, incubate at 4°C overnight, let stand at room temperature for 1 hour, wash with PBS 2-3 times, 8-12 minutes each time; add secondary antibody, incubate at room temperature in the dark for 1.5-2.5 hours, wash with PBS 2-3 times, 10 minutes each time;

[0093] Add DAPI and incubate for 10 min, wash 2-3 times with PBS, 55 min each time, and seal the slides;

[0094] D. Observation and calculation: Observe and take pictures under a fluorescence microscope. The cell purity is the ratio of the number of cells stained with GFAP to the number of cells stained with DAPI.

[0095] Specifically, constructing the miR-31 overexpression astrocyte model in S2 includes the following steps:

[0096] (1) The prepared astrocytes were seeded in a six-well plate and then incubated in a culture medium containing 10% FBS and 1% double antibody for 8-16 h in an incubator;

[0097] (2) diluting the transfection reagent, pipetting 3-5 times to mix, diluting the siRNA, pipetting 3-5 times to mix and letting stand for 3-8 min, mixing the two liquids, pipetting 3-5 times to mix and letting stand at room temperature for 15-25 min to obtain a mixed reagent;

[0098] (3) Remove the cells from the incubator, wash the cells 2-3 times with PBS, and then remove the residual PBS. Add 1500 μL of culture medium containing 12% FBS without dual antibodies to each well, drop the mixed reagent obtained in (2) into the well, shake the plate gently to mix, and replace the complete culture medium containing dual antibodies after 3-6 hours in the incubator. Perform subsequent operations after 12-24 hours.

[0099] Specifically, in step (2), every 6 μL of Lipofectamine 2000 transfection reagent was diluted with 250 μL of Opti-Mem, and every 5 μL of siRNA was diluted with 250 μL of Opti-Mem.

[0100] Specifically, in S4, the molecular level validation verified the miR-31 regulatory mechanism through gene expression analysis, sequencing, and luciferase reporter gene assay, and the functional validation evaluated the functional effect of miR-31 through cell proliferation experiments and protein expression analysis.

[0101] The present invention provides the use of miR-31 in preparing a medicine or composition for a spinal cord injury repair process.

[0102] The present invention provides the use of miR-31 in preparing a medicine or a composition for treating a neurodegenerative disease caused by spinal cord injury.

[0103] Example 1

[0104] Isolation, culture and purification of astrocytes

[0105] 1. Extraction and purification of primary astrocytes

[0106] (1) Preparation in advance: high-pressure sterilized scissors and tweezers; poly-lysine-coated culture bottles; because multiple newborn mice are needed, they should be caged together in advance (one male and three females per cage).

[0107] (2) Take 2-3 day old newborn FVB mice, disinfect them with iodine, and place them in a small dish placed on an ice box (pre-cooled D-hank solution is placed in the small dish in advance). Decapitate and cut open the dorsal side near the tail end, remove the skin and muscles covering the spine, separate the muscles on both sides of the spine, and peel off the cervical to lumbar vertebrae to obtain a complete spine.

[0108] (3) Place the excised spinal cord into a new D-hank, remove the larger blood vessels and adventitia, rinse three times, and then cut into pieces.

[0109] (4) In 0.25% trypsin-EDTA, pipette for 30 seconds, place on a shaker for digestion for 20 minutes, add 2 mL of complete culture medium containing 10% FBS and 1% double antibody to terminate digestion, pass through a nylon sieve to remove larger tissues, and collect the precipitate after centrifugation for 5 minutes.

[0110] (5) Resuspend the cells in complete culture medium containing 10% FBS and 1% dual antibody, inoculate them into the culture flask coated in advance, and transfer them to a new culture flask after 30 minutes of differential adhesion.

[0111] (6) After 10-14 days when the confluence reaches 80%, the cells are purified on a shaker (180 rpm, 20 h) to remove microglia and oligodendrocytes, and then digested and passaged.

[0112] 2. Passaging

[0113] Take out the cells from the incubator, observe under the microscope that the cells are growing well and the confluence reaches 80%, then pass them on, wash twice with PBS, add 2-3mL of trypsin, put the culture bottle into the incubator for digestion for 3 minutes, then take it out, observe under the microscope whether the cells have become round, tap the bottom of the bottle when the cells are firmly attached to the bottom of the bottle, add 2mL of complete culture medium containing 10% FBS and 1% double antibody to stop digestion, blow and beat so that the cells can be completely collected into a 15mL centrifuge tube, centrifuge, discard the supernatant, and take the precipitate. Add 5mL of culture medium to the precipitate and gently resuspend it, evenly spread it into a new culture bottle, and observe the cell status in time.

[0114] 3. Cryopreservation and Thawing

[0115] Cryopreservation: Prepare the cryopreservation solution first: 55% basal medium + 40% FBS + 5% DMSO (cell grade). When the cells grow to 90% confluence, wash twice with PBS, add 2mL pancreatin, shake gently, put into the incubator for 3 minutes, take out, observe the cells become round under the microscope, and terminate the digestion with complete medium. Puff the cells, collect them into a 15mL centrifuge tube, centrifuge, and discard the supernatant. Resuspend the cells with the pre-prepared cryopreservation solution, put them into the cryopreservation tube, and put them into the -80℃ refrigerator.

[0116] Recovery: After taking out the cells from the -80℃ refrigerator or liquid nitrogen, immediately place them in a 37℃ water bath. After thawing for 2 minutes, immediately pour the liquid into a centrifuge tube filled with complete culture medium and mix well by pipetting. Centrifuge, discard the supernatant, resuspend the cell pellet with complete culture medium, and place it in a culture bottle.

[0117] Example 2

[0118] Immunofluorescence staining of cells to identify astrocyte markers and detect cell purity

[0119] (1) Preparation in advance: Soak the coverslip in 75% alcohol overnight, dry it with an alcohol lamp and place it in a six-well plate; prepare poly-L-lysine (0.1 mg / mL) for later use;

[0120] Drop the prepared poly-lysine on each coverslip, put it in the incubator, aspirate it after 4 hours, wash it 3 times with PBS, put it in the incubator to dry overnight for use: prepare 0.5% TritonX-100; dilute the antibody according to the instructions and package it; prepare BSA.

[0121] (2) The day before: Resuspend the cells on coverslips, 3×10 cells per well 5 The cells were placed in an incubator overnight;

[0122] (3) Take out the six-well plate from the incubator, discard the culture medium, and wash twice with PBS; fix the slides with 4% paraformaldehyde for 30 min and wash twice with PBS;

[0123] (4) Add 200 μL TritonX-100 to each well, permeabilize for 20 min at room temperature, and wash three times with PBS for 3 min each;

[0124] (5) Lift the slide, dry it and put it back into the six-well plate; add 1 mL BSA, block at room temperature for 1 h, and wash twice with PBS;

[0125] (6) Place the slide on a glass slide, drop 100 μL of primary antibody, place in a wet box, and incubate at 4°C overnight; place at room temperature for 1 hour until it reaches room temperature, and wash with PBS three times, 10 minutes each time;

[0126] (7) Add secondary antibody and incubate at room temperature in the dark for 2 h; wash with PBS three times, once for 10 min;

[0127] (8) Add DAPI and incubate for 10 min, wash with PBS 3 times, 5 min each time, and seal the slides, making sure there are no bubbles.

[0128] (9) Observe and photograph under a fluorescence microscope. The cell purity is calculated by dividing the number of cells stained with GFAP by the number of cells stained with DAPI.

[0129] Example 3

[0130] Construction of miR-31 transfected astrocyte model

[0131] (1) One day before: Add 7 × 10 5 The cells were incubated in a culture medium containing 10% FBS and 1% double antibody overnight in an incubator.

[0132] (2) Dilute 6 μL Lipofectamine 2000 transfection reagent with 250 μL Opti-Mem and pipette 3-5 times to mix well;

[0133] Dilute 5 μL siRNA with 250 μL Opti-Mem, pipette 3-5 times to mix, and let stand for 5 min; mix the two liquids, pipette 3-5 times to mix, and let stand at room temperature for 20 min.

[0134] (3) Remove the cells from the incubator, wash them twice with PBS, and then aspirate them clean. Add 1500 μL of medium containing 12% FBS without dual antibodies to each well. Drop the mixed reagent into the well and gently shake the plate to mix. After 5 hours in the incubator, replace the complete medium containing dual antibodies. Perform subsequent experiments 24 hours later.

[0135] Example 4 Real-time fluorescence quantitative PCR to verify transfection efficiency

[0136] 1. RT-qPCR verification of transfection efficiency

[0137] (1) RNA extraction (Trizol extraction) Discard the culture medium, wash twice with PBS, add 1 ml of Trizol, lyse and transfer to an EP tube, and incubate for 10 min.

[0138] Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes; centrifuge at 10,000 g for 15 minutes.

[0139] After centrifugation, separate into three layers. Transfer the upper colorless layer to a new centrifuge tube, add 500 μL of isopropanol, mix well, let stand for 5 minutes, and then centrifuge at 10000g for 10 minutes. At this time, a precipitate is formed at the bottom of the tube, and the supernatant is discarded.

[0140] Wash twice with 75% ethanol, discard the supernatant, dry, and dissolve the precipitate in 20 μL DEPC water. (2) Reverse transcription to cDNA. The reaction system of Takara's miRNA-specific reverse transcription reagent is as follows:

[0141]

[0142] Set the thermal cycle to 37°C for 1 h; 85°C for 5 min.

[0143] (3) RT-qPCR stage

[0144] Design primers, the primer sequence numbers are as follows:

[0145]

[0146] Samples were added to eight rows according to the groups. Each group required 3 replicates. Each target gene required a corresponding internal reference U6. The reaction system of U6 and miR-31 was shown below.

[0147]

[0148]

[0149] Calculation results after on-machine testing

[0150] Denaturation stage: 95℃ for 10s;

[0151] qPCRx40Cycles stage: 95℃5s; 60℃20s;

[0152] DissociationCurve stage: 95℃60s; 55℃30s; 95℃30s.

[0153] 2. CCK-8 detection of cell proliferation

[0154] (1) After the cells are transfected, they are divided into different 96-well plates according to different time points. PBS is added to the outermost circle of each plate to prevent evaporation and marked;

[0155] (2) Calculate the total number of cells required for each group based on the digestion and centrifugation counts, and take the corresponding cell suspension into a 15 ml centrifuge tube and add it to a 96-well plate, with 100 μL of cell suspension in each well;

[0156] (3) At the time point, take out the plate, discard the culture medium and add the prepared CCK-8 detection solution. The detection solution in each well is 10 μL CCK-8 detection solution and 90 μL culture medium. Place in the incubator and incubate for 2 hours. Detect the absorbance at 450 nm on the microscope.

[0157] 3. Statistical Analysis

[0158] Statistical analysis was performed using statistical analysis software (SPSS Statistics 26.0, IBM, USA), and data were plotted using graphics software (Prism Graph Pad 9.0, Graph Pad Software, USA). All experimental quantitative data were tested for normality and variance homogeneity using the Shapiro-Wilk test and Levene test, respectively, and all data met normal distribution and variance homogeneity. One-way analysis of variance and independent sample t-test were used to analyze the differences between groups for all quantitative data. The significance level of all tests was set to two-sided a = 0.05, and P < 0.05 indicated that the difference was statistically significant.

[0159] Example 5 Molecular level verification

[0160] Materials and Reagents:

[0161] instrument

[0162]

[0163] Material

[0164]

[0165] 1. Cell Transfection and Total RNA Extraction

[0166] The astrocytes extracted in the first part were subcultured to the third generation. After 72 hours of transfection, the cells were lysed by Trizol method to extract RNA. The samples were dissolved in DEPC to obtain total RNA. The RNA purity and concentration were tested on a machine and then transcriptome sequencing was performed.

[0167] 2. Construction of sequencing library and on-machine detection

[0168] The RNA samples were enriched with polyA to generate fragmented RNA sequences, which were then reverse transcribed into double-stranded cDNAs, and "Y"-shaped adapters were connected to both ends of the double-stranded cDNAs to form a standard sequencing library. After PCR amplification, sequencing was performed using the Novasek platform (sequencing work was commissioned to Novogene Technology Co., Ltd. to assist).

[0169] 3. Analysis of Sequencing Results

[0170] The data obtained by sequencing were merged and analyzed after passing the quality inspection. After comparing the genes that met the expected expression level between the groups, GO and KEGG enrichment analysis was performed, and the screened DEGs were integrated and analyzed according to function and signal pathway to find genes and signal pathways related to cell proliferation. Finally, based on the Targetscan website, it was determined whether the DEGs that met the requirements had binding sites with miR-31, and its downstream target genes were screened accordingly.

[0171] Example 6 Dual luciferase reporter gene assay

[0172] (1) The 3'UTR of LATS2 was constructed into the pmirGLO vector, and the recombinant plasmids were named LATS2WT-mmu-miR-31-5p and LATS2 MUT-mmu-miR-31-5p, respectively (commissioned by Gene Pharma).

[0173] (2) Use the Biosharp Endotoxin Removal Kit to extract the plasmid, the steps are as follows:

[0174] A. Add 2.5 mL of equilibration solution to the adsorption column, centrifuge at 10,000 rpm for 2 min, discard the filtrate, and put the adsorption column back into the centrifuge tube; take 200 mL of strain, centrifuge at 10,000 rpm for 2 min, and discard the supernatant.

[0175] B. Add 10 mL of Buffer I and vortex to mix evenly, showing a turbid brown-red color; add 10 mL of Buffer II and gently invert to mix until the solution turns into a cool, viscous purple-red color.

[0176] C. Add 10 mL of Buffer III and gently invert to mix until it turns completely yellow. Centrifuge at 10,000 rpm for 10 min, transfer the supernatant to a new centrifuge tube, and add 0.3 times the volume of isopropanol in the filtrate and invert to mix.

[0177] D. Transfer the liquid to the centrifuge tube in the first step, centrifuge at 10,000 rpm for 1 min, and discard the filtrate. Add 5 mL Endotoxin Buffer, let stand for 5 min, centrifuge at 10,000 rpm for 1 min, and discard the filtrate.

[0178] E. Add 10 mL of Buffer W1, centrifuge at 10,000 rpm for 1 min, discard the filtrate, repeat twice, add 1 mL of elution buffer, and centrifuge to obtain the plasmid.

[0179] (3) Cell treatment

[0180] Inoculate cells in a 24-well plate and divide them into four groups for transfection, and add a blank control group (NC). 24 hours after transfection, aspirate the culture medium, add 200 μL lysis solution, and gently rotate the culture plate to cover the cells with lysis solution. Incubate on ice for 5 minutes, centrifuge at 10000 rpm for 1 minute, and take the supernatant.

[0181] (4) Fluorescence detection

[0182] A. Place 20 μL of lysate into each well of a 96-well culture plate.

[0183] B. Dilute 50× firefly and Renilla luciferase substrates to 1× working solution with corresponding buffer and return to room temperature.

[0184] C. Add 100 μL of firefly luciferase reaction solution, shake and mix, and then detect the activity (30 minutes to complete).

[0185] D. Add 100 μL of Renilla luciferase reaction solution, shake and mix, and then detect the activity (30 minutes to complete).

[0186] (5) Analyze data

[0187] Background F: NC + firefly luciferase reaction solution;

[0188] Background R: NC + firefly luciferase reaction solution + Renilla luciferase reaction solution;

[0189] Experimental group: miR-31mimics group was treated as above;

[0190] Control group: miR-31mimics NC group was treated as above;

[0191] Experimental group ratio = (experimental group F-background F) / (experimental group R-background R)

[0192] The ratio of the control group is the same as above

[0193] Expression multiple = experimental group ratio / control group ratio

[0194] RT-qPCR

[0195]

[0196] The experimental steps are the same as above.

[0197] Example 7 Flow cytometry

[0198] Flow cytometry to measure cell proliferation

[0199] 1) Seed the plates, transfect, and conduct the experiment 24 hours later.

[0200] 2) Wash twice with PBS, digest with trypsin, blow the cells, remove the supernatant by centrifugation, and collect the cell pellet in an EP tube.

[0201] 3) Add 50 μM EdU and incubate for 2 h to incorporate EdU into the cells. Wash the cells with 1 mL 1% BSA, centrifuge at 2000 rpm for 3 min at room temperature, and remove the supernatant.

[0202] 4) Resuspend the cells with 4% paraformaldehyde, incubate at room temperature in the dark for 15 min, wash the cells twice with 1 mL 1% BSA, centrifuge at 2000 rpm for 3 min at room temperature, and remove the supernatant.

[0203] 5) Resuspend the cells with 500 μL 0.5% Triton X-100, incubate for 20 min, centrifuge at 2000 rpm for 3 min at room temperature, and wash the cells twice with 1% BSA, each time for 5 min.

[0204] 6) Prepare 5× Click-iT EdU reaction additive stock solution and dilute a certain amount to 1× for use in the experiment.

[0205] 7) Prepare the reaction mixture according to the table below. Add 1 mL of reaction mixture to each well of the six-well plate.

[0206]

[0207] 8) Incubate at room temperature in the dark for 30 min, wash the cells once with 1 mL of 0.5% Triton X-100, centrifuge, remove the supernatant, resuspend the cells with 1% BSA, and measure the value at 488 nm.

[0208] Example 8 Western blot analysis

[0209] Primary antibodies: LATS2 (dilution ratio 1:400, AB clonal), YAP (dilution ratio 1:1000, Cell Signaling Technology), β-actin (dilution ratio 1:400, AB clonal);

[0210] Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (dilution ratio 1:6000, ABclonal);

[0211] (1) Protein extraction

[0212] Prepare ice, add 1 / 100 PMSF, protease inhibitors and phosphatase inhibitors to WB Lysis Buffer, and place on ice for later use. Remove cells, discard culture medium, add 100 μL of prepared working solution to each well, shake on ice for 10 minutes, ultrasonically shake, scrape cells after full lysis, centrifuge at 14000g for 5 minutes, take supernatant, and perform subsequent concentration determination.

[0213] (2) BCA method for measuring protein concentration

[0214] A. Prepare BSA standard according to the instructions and make a standard curve.

[0215] B. Prepare BCA working solution, A:B=50:1, dilute the protein 2-5 times, add 200μL BCA working solution and 20μL test protein to each well, incubate at 37℃ for 30min, detect the absorbance at 562nm, and calculate the protein concentration.

[0216] C. After adding buffer, boil in metal bath at 100℃ for 10 min and store at -80℃.

[0217] (3) Glue preparation

[0218] A. Clean the glass plate, align the bottom of the long and short plates on the table and clamp them with glue clamps. After clamping, clamp them with glue racks, and make sure to put glue pads underneath. Use triple distilled water to check for leaks, and use filter paper to absorb moisture.

[0219] B. Prepare the lower layer of 10% separation gel according to the table below, mix well and add it between the long and short plates, press it flat with anhydrous ethanol, let it stand for 50 minutes, and after the separation gel is fully solidified, pour out the upper layer of anhydrous ethanol and absorb it dry with filter paper.

[0220]

[0221] C. Prepare 5% upper concentrated gel according to the table below, mix well and add to the upper space of the glass plate, insert a comb, and store at 4°C after solidification.

[0222]

[0223] (4) Electrophoresis

[0224] A. Put the gel and plastic plate into the electrophoresis apparatus, clamp them, and put them into the electrophoresis tank. Fill the middle of the electrophoresis apparatus with electrophoresis buffer and add the sample slowly.

[0225] B. Add electrophoresis buffer into the electrophoresis tank, connect the electrodes, and turn on the power.

[0226] C. The initial parameters are set to 80V for 1h, and after the marker is separated, the parameters are reset to 120V for 30min.

[0227] (5) Transfer

[0228] A. Take out the gel after electrophoresis, cut off the excess part and put it into the pre-cooled electrotransfer solution, cut the filter paper and PVDF membrane according to the size of the gel, mark the PVDF membrane and put it into methanol for activation for 3 minutes; fix the filter paper, gel and sponge in order and put them into the electrotransfer tank.

[0229] B. Set the transfer parameters to 250mA for 1.5h.

[0230] (6) Blocking and incubation with antibodies

[0231] After transfer, the membrane was removed, blocked with 10% skim milk powder at room temperature on a shaker for 2 h, washed five times with TBST for 5 min each time, added with diluted primary antibody, and incubated at 4°C on a shaker overnight; the primary antibody was recovered the next day, washed five times with TBST for 5 min each time, and then incubated with diluted secondary antibody at room temperature on a shaker for 1 h. After that, the secondary antibody was recovered and washed five times with TBST for 5 min each time.

[0232] (7) Development.

[0233] Statistical analysis

[0234] Statistical analysis was performed using statistical analysis software (SPSS Statistics 26.0, IBM, USA), and data were plotted using graphing software (Prism GraphPad 9.0, GraphPad Software, USA). All experimental quantitative data were tested for normality and variance homogeneity using the Shapiro-Wilk test and Levene test, respectively, and all data met normal distribution and variance homogeneity. One-way analysis of variance and independent sample t-test were used to analyze the differences between groups for all quantitative data. The significance level of all tests was set to two-sided a=0.05, and P<0.05 indicated that the difference was statistically significant.

[0235] Example 9

[0236] 1. Analysis of differentially expressed genes of MiR-31

[0237] The miR-31 overexpression group and the control group, as well as the miR-31 inhibitor group and the control group were compared, and the obtained DEGs were plotted into cluster volcano maps using the R language ggplot2 package and pheatmap package. The results showed that there were 1283 significantly upregulated DEGs and 1208 significantly downregulated DEGs in the miR-31 overexpression group; there were 1138 significantly upregulated DEGs and 870 significantly downregulated DEGs in the miR-31 inhibitor group (e.g. Figure 1 MiR-31 regulates target gene expression by destroying the stability of target mRNA or inhibiting its translation. Therefore, it is necessary to screen out the DEGs that are downregulated in the miR-31 overexpression group and upregulated in the miR-31 inhibitor group, and draw a Venn diagram for the screened data. Among them, 297 DEGs met the expression expectations (as shown in Figure 2 shown).

[0238] 2GO and KEGG enrichment analysis of differentially expressed genes

[0239] GO enrichment analysis was performed on the 297 potential target genes screened. The results showed that the proportion of DEGs in biological processes such as regulation of cell size and metabolism, DNA metabolism, dendrite development and morphogenesis, molecular functions such as endoplasmic reticulum membrane, lysosome, Golgi network, and nucleotide and purine nucleoside binding was high (e.g. Figure 3 ).

[0240] The KEGG database is a database that analyzes gene function and links genomic information and functional information. It is the most widely used public database of metabolic pathways. The KEGG analysis results of DEGs showed that they were mainly enriched in signaling pathways such as Rap1, Ras, and Hippo (see Figure 4 ). The Hippo signaling pathway, also known as the Salvador / Warts / Hippo (SWH) pathway, is named after the protein kinase Hippo in Drosophila and is a key regulator in the pathway. This pathway is composed of a series of conserved kinases and is one of the main signaling pathways regulating cell proliferation and apoptosis in multicellular animals. It plays a key role in maintaining tissue homeostasis and regulating organ size.

[0241] 3. Validation of potential target genes of MiR-31

[0242] The target genes of mmu-miR-31-5p were predicted by Targetscan database, and combined with GO and KEGG enrichment results for comprehensive analysis, it was found that the kinase LATS2 of Hippo signaling pathway may be the target gene of mmu-miR-31-5p, and its 3'UTR region has a binding site for mmu-miR-31-5p (see Figure 5 A). The expression of LATS2 after transfection was verified by RT-qPCR, and it was found that miR-31 was negatively correlated with LATS2 expression. As the drug concentration increased, the expression of LATS2 in the miR-31mimics group gradually decreased compared with the NC group. When the drug concentration was 500nM, the expression of LATS2 in the miR-31mimics group reached the lowest level (see Figure 5 B, P<0.001), while the expression of LATS2 in the miR-31inhibitor group reached the highest level when the drug concentration was 300 nM (see Figure 5 C, P<0.001). The above results are consistent with the expected expression trend of target genes, which suggests that mmu-miR-31-5p may target LATS2 to promote astrocyte proliferation.

[0243] 4. Verification of the specific binding of miR-31-5p to LATS2 3'UTR

[0244] To further prove that LATS2 is a direct target gene of miR-31, a dual luciferase reporter gene assay was performed. In this example, a dual luciferase reporter gene vector of LATS2 3'UTR and a dual luciferase reporter gene vector of a mutant 3'UTR with site-directed mutation of the miR-31 binding site were constructed (mutation information is shown in Figure 6 ). After co-transfection of mmu-miR-31-5pmimics with plasmid into astrocytes for 24 h, the dual luciferase activity was detected and the ratio of the two luciferase activities was calculated. The results showed that the ratio of firefly luciferase / sea renilla luciferase in the WT+miR-31mimics group was significantly lower than that in the WT+miR-31mimics NC group (P < 0.05), while miR-31mimics had no significant effect on the mutant vector (P > 0.05, as shown in Figure 2). Figure 7 ). This indicates that mmu-miR-31-5p can directly bind to the LATS2 3'UTR region and specifically inhibit the expression of firefly luciferase.

[0245] Effects of 5 target genes on astrocyte proliferation

[0246] LATS2 is a target gene product of miR-31, but its biological function in astrocytes remains unknown. To explore the effect of LATS2 on astrocyte proliferation, RT-qPCR experiments were first performed to verify that the expression of LATS2 was knocked down 5-fold at the mRNA level (named siRNA-LATS2, see Figure 8 A, P < 0.001) and increased by more than 600 times (named cDNA-pLats2, see Figure 8 B, P<0.001). EdU is a pyrimidine analog that can be incorporated into the DNA double strand during DNA synthesis and is one of the most accurate methods for detecting cell proliferation. The EdU ratio of each group was detected by flow cytometry to measure cell proliferation, which further confirmed the inhibitory effect of LATS2 on astrocyte proliferation (see Figure 8 C, P<0.05).

[0247] 6. Relative expression of target gene protein

[0248] The relative expression of LATS2 and YAP proteins was then analyzed by Western blot. The experimental results showed that the relative expression of LATS2 protein in the miR-31 mimics group was significantly lower than that in the NC group (P<0.05, see Fig. 9 AB), while the relative expression of LATS2 protein in the miR-31inhibitor group was significantly higher than that in the NC group (P<0.05, Fig. 9 AB), further confirming that LATS2 is a target protein of miR-31. In this example, it was found that the motor function of mice with miR-31 overexpression after spinal cord injury was improved compared with that of wild-type FVB mice. On this basis, the preliminary mechanism of the regulatory effect of intrinsic overexpression of miR-31 was explored (see Fig.10 ), found that miR-31 promoted astrocyte proliferation by targeting the Hippo pathway kinase LATS2, thereby promoting tissue repair after spinal cord injury.

[0249] in conclusion:

[0250] After breeding and identifying miR-31 overexpression positive mice, this application constructed spinal cord injury models of miR-31 overexpression positive mice and wild-type FVB mice, respectively. The BMS score and gait analysis results showed that the motor function recovery of miR-31 overexpression positive mice after spinal cord injury was better than that of wild-type FVB mice, and further explored its possible intrinsic regulatory role.

[0251] This application extracted high-purity primary astrocytes by enzyme digestion, and then overexpressed miR-31 in astrocytes by transfection, and found that miR-31 overexpression can promote astrocyte proliferation. Finally, RNA-seq was used to obtain data on possible downstream targets of miR-31 regulating astrocyte proliferation. By comparing DEGs, GO and KEGG enrichment analysis between groups, the Hippo pathway closely related to cell proliferation was determined. Combined with the target gene prediction website, the Hippo pathway kinase LATS2 was screened as a possible downstream target gene, and further verified by dual luciferase reporter gene assay, RT-qPCR, Western blot and flow cytometry, providing a theoretical basis for the clinical treatment of spinal cord injury.

[0252] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A target gene for miR-31 regulating astrocyte proliferation, characterized in that: The key downstream target gene of miR-31 in regulating astrocyte proliferation is LATS2. The sequence of miR-31 is shown in SEQ ID NO.1, and the sequence of LATS2 is shown in SEQ ID NO.

2.

2. A method for screening target genes for miR-31 regulating astrocyte proliferation, characterized in that: The following steps are involved: S1. Isolation of astrocytes: Isolate and culture astrocytes, identify astrocyte markers and detect cell purity by immunofluorescence; S2. Establish a miR-31 overexpression model: Genetically engineer and identify miR-31 overexpression positive mice, and construct a miR-31 overexpression astrocyte model at the cellular level by transfection technology; S3. Real-time fluorescence quantitative PCR to verify transfection efficiency: RT-qPCR and CCK-8 experiments were used to verify the transfection efficiency of miR-31 and its effect on cell proliferation; S4. Molecular level verification and functional verification.

3. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 2, characterized in that: The specific marker of astrocytes is GFAP, and the GFAP is combined with DAPI to label astrocytes to identify and confirm the purity of astrocytes.

4. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 2, characterized in that: The isolation, culture and purification of astrocytes in S1 comprises the following steps: a. Extraction and purification of primary astrocytes: Spinal cord tissue was isolated from 2-3 day old FVB mice, and high-purity primary astrocytes were obtained through digestion, purification and culture; b. Passaging: After confirming that the cells are growing well and the confluence reaches 80% under a microscope, wash them twice with PBS, add trypsin to digest for 3-5 minutes, terminate the digestion with complete culture medium, collect the cells by centrifugation, resuspend them, and inoculate them into a new culture flask; c. Cryopreservation and thawing: After washing the cells with a confluence of 90% twice with PBS, add 2 mL of trypsin to digest for 3 minutes. After the cells become round under the microscope, stop digestion with complete medium, collect the cells by centrifugation and discard the supernatant, freeze them at -80°C with the cryopreservation solution prepared in advance, and inoculate them into culture flasks after rapid thawing and resuspending the cells with complete medium when needed; After taking out the cells from the -80℃ freezer or liquid nitrogen, immediately place them in a 37℃ water bath to thaw for 2 minutes, then transfer the cell suspension to a centrifuge tube containing complete culture medium and mix well. After centrifugation, discard the supernatant, resuspend the cells in complete culture medium and inoculate them into the culture flask.

5. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 4, characterized in that: The immunofluorescence method for identifying cell purity in S1 includes the following steps: A. Preliminary preparation: Soak the coverslip in 75% alcohol overnight, dry it with an alcohol lamp and place it in a six-well plate, prepare a 0.1 mg / mL poly-lysine solution, drop it on the coverslip, place it in an incubator for 4 hours, then aspirate it, wash it 3 times with PBS, place it in an incubator to dry overnight, and prepare BSA; B. Cell culture and fixation: The day before, resuspend the cells on coverslips at 3 × 10 cells per well. 5 The cells were placed in an incubator overnight, and the six-well plate was removed from the incubator, the culture medium was discarded, and the plate was washed twice with PBS, fixed with 4% paraformaldehyde for 20-40 min, and washed 2-3 times with PBS; C. Immunofluorescence staining: Add 200 μL TritonX-100 to each well, permeabilize for 20 min at room temperature, and wash 2-3 times with PBS, each time for 2-5 min; Add 1 mL BSA to block for 1 h, and wash 2-3 times with PBS; Add primary antibody, incubate at 4°C overnight, let stand at room temperature for 1 hour, wash with PBS 2-3 times, 8-12 minutes each time; add secondary antibody, incubate at room temperature in the dark for 1.5-2.5 hours, wash with PBS 2-3 times, 10 minutes each time; Add DAPI and incubate for 10 min, wash 2-3 times with PBS, 55 min each time, and seal the slides; D. Observation and calculation: Observe and take pictures under a fluorescence microscope. The cell purity is the ratio of the number of cells stained with GFAP to the number of cells stained with DAPI.

6. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 2, characterized in that: The construction of the miR-31 overexpression astrocyte model in S2 includes the following steps: (1) The prepared astrocytes were seeded in a six-well plate and then incubated in a culture medium containing 10% FBS and 1% double antibody for 8-16 h in an incubator; (2) diluting the transfection reagent, pipetting 3-5 times to mix, diluting the siRNA, pipetting 3-5 times to mix and letting stand for 3-8 min, mixing the two liquids, pipetting 3-5 times to mix and letting stand at room temperature for 15-25 min to obtain a mixed reagent; (3) Remove the cells from the incubator, wash the cells 2-3 times with PBS, and then remove the residual PBS. Add 1500 μL of culture medium containing 12% FBS without dual antibodies to each well, drop the mixed reagent obtained in (2) into the well, shake the plate gently to mix, and replace the complete culture medium containing dual antibodies after 3-6 hours in the incubator. Perform subsequent operations after 12-24 hours.

7. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 6, characterized in that: In the step (2), every 6 μL of Lipofectamine 2000 transfection reagent was diluted with 250 μL of Opti-Mem, and every 5 μL of siRNA was diluted with 250 μL of Opti-Mem.

8. The method for screening target genes for miR-31 regulating astrocyte proliferation according to claim 2, characterized in that: The molecular level verification in S4 verifies the miR-31 regulatory mechanism through gene expression analysis, sequencing, and luciferase reporter gene assay, and the functional verification evaluates the functional effect of miR-31 through cell proliferation experiment and protein expression analysis.

9. Application of miR-31 in the preparation of drugs or compositions for spinal cord injury repair process.

10. Use of miR-31 in the preparation of a drug or composition for treating neurodegenerative diseases caused by spinal cord injury.

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