WO2022110651A1

WO2022110651A1 - Mir-30a-5p and use thereof in promoting nerve regeneration and repairing peripheral nerve injury

Info

Publication number: WO2022110651A1
Application number: PCT/CN2021/092308
Authority: WO
Inventors: 丁斐; 周松林; 施海燕; 从猛; 沈宓; 张琦
Original assignee: 南通大学
Priority date: 2020-11-24
Filing date: 2021-05-08
Publication date: 2022-06-02
Also published as: CN112301033A; CN112301033B; AU2021307014B2; AU2021307014A1

Abstract

A miR-30a-5p and a use thereof in promoting nerve regeneration and repairing peripheral nerve injury. The miR-30a-5p is a molecular target for repairing peripheral nerve injury, and is used to prepare a drug for promoting nerve regeneration and repairing nerve injury. The use thereof in promoting DRG neuron axon regeneration and repairing peripheral nerve injury comprises the following verification steps: S1, culturing primary DRG neuron cells, and observing how in vitro overexpression of the miR-30a-5p promotes DRG neuron axon growth; S2, culturing primary DRG neuron cells, and observing how in vitro overexpression of the miR-30a-5p promotes DRG neuron axon regeneration; S3, extracting primary DRG neuron cells, and observing how in vitro overexpression of the miR-30a-5p inhibits NRP1mRNA and protein expression. By using the miR-30a-5p as a molecular intervention target and overexpressing the miR-30a-5p, the growth and regeneration of DRG neuron axons are promoted. By culturing in vitro DRG neurons using microfluidics, and transfecting a miR-30a-5p mimic, the growth and regeneration of the primary cultured DRG neuron axons may be significantly promoted.

Description

miR-30a-5p and its application in promoting nerve regeneration and repairing peripheral nerve injury

technical field

The invention belongs to the technical field of biomedicine, in particular to a miR-30a-5p and its application in promoting nerve regeneration and repairing peripheral nerve damage.

Background technique

The repair of peripheral nerve injury is a common clinical problem, which causes a great burden to the society and family. Peripheral nerves can regenerate spontaneously after injury, but due to the limited regeneration rate, it is ultimately difficult to restore function. Therefore, fully exploring the cellular and molecular mechanisms of peripheral nerve injury regeneration will help to promote peripheral nerve function repair and provide a theoretical basis for clinical treatment.

microRNA (miRNA) is an endogenous non-coding small RNA with a length of about 21-23 nucleotides. Its main role is to inhibit the translation of target genes or Direct degradation of target genes. In the peripheral nervous system, miRNA can not only inhibit the apoptosis of neurons and promote the regeneration of neuronal axons, but also participate in the regulation of the proliferation and migration of glial cells. Studies have found that DRG neuron axon growth is helpful for the repair of peripheral nerve damage. Therefore, it is necessary to provide a new target that promotes the growth of DRG neuron axons and is helpful for the treatment of nerve injury.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a miR-30a-5p and its application in promoting nerve regeneration and repairing peripheral nerve damage. Overexpression of miR-30a-5p in vitro can target Nrp1 and significantly promote the DRG neuron axis The growth and regeneration of neurites provide a new target for the repair of peripheral nerve injury.

In order to solve the above technical problems, the embodiments of the present invention provide a miR-30a-5p, which is a molecular target for peripheral nerve damage repair.

The invention also provides a use of miR-30a-5p for preparing a drug for promoting nerve regeneration and repairing nerve damage.

Wherein, the nerve injury is a peripheral nervous system sciatic nerve injury.

The present invention also provides an application of miR-30a-5p in promoting DRG neuron axon regeneration and peripheral nerve damage repair, including the following verification steps:

S1. Cultivate primary DRG neuron cells, and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon growth;

S2. Cultivate primary DRG neuron cells, and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon regeneration;

S3. Extract primary DRG neuron cells, and observe the inhibition of NRP1 mRNA and protein expression by overexpression of miR-30a-5p in vitro.

Wherein, the specific steps of step S1 are:

S1.1. Microfluidic culture of primary DRG neuron cells

S1.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with 3mg/ml collagenase, 37°C, 30min;

S1.1.2. Discard collagenase, add 0.25% trypsin to digest, 37℃, 20min;

S1.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S1.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with poly-lysine. After culturing for 4 hours, replace them with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine for depletion of non-neuronal cells;

S1.2. Transfection of neuronal cells with mimics

After the cells adhered, miR-30a-5p mimics and negative control were mixed with transfection reagent, added to the neuron cells cultured in step S1.1, and replaced with neuron medium after 8 hours of culture;

S1.3. Cell immunofluorescence staining and axonal growth length measurement

S1.3.1. Discard the cell culture medium after 72h of DRG neuron transfection, rinse with PBS, add 4% paraformaldehyde, and fix for 30min;

S1.3.2. After discarding the paraformaldehyde, wash with PBS three times for 5 minutes each time;

S1.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

S1.3.4. Dilute the primary antibody anti-Tuj1 antibody (1:400, abcam) with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

S1.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

S1.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG (1:400, Sigma) with immunohistochemical secondary antibody diluent, add the secondary antibody, and keep it at room temperature for 2 hours in the dark;

S1.3.7. Discard the secondary antibody, wash 3 times with PBS, 5min each time;

S1.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the axonal growth of DRG neurons, take pictures and count the distribution of neurite lengths in each group.

Wherein, the specific steps of step S2 are:

S2.1. Microfluidic culture of primary DRG neuron cells

S2.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with 3mg/ml collagenase, 37°C, 30min;

S2.1.2. Discard collagenase, add 0.25% trypsin to digest, 37℃, 20min;

S2.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S2.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with polylysine. After culturing for 4 hours, replace them with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine for depletion of non-neuronal cells;

S2.2. Transfection of neuronal cells with mimics

After the cells adhered, the miR-30a-5p mimics and negative control were mixed with transfection reagent and added to the cultured neurons, and the culture medium was replaced after 8 hours of culture;

S2.3. Cell immunofluorescence staining and axon regeneration length measurement

S2.3.1. After 72 hours of transfection of DRG neuron cells, the axons of the cells in the chamber were removed by negative pressure. After 24 hours, the cell culture medium was discarded, rinsed with PBS, and 4% paraformaldehyde was added. Fixed for 30min;

S2.3.2. After discarding the paraformaldehyde, wash with PBS three times for 5 minutes each time;

S2.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

S2.3.4. Dilute the primary antibody anti-Tuj1 antibody with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

S2.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

S2.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG with immunohistochemical secondary antibody diluent, add the secondary antibody, and protect from light at room temperature for 2 hours;

S2.3.7. Discard the secondary antibody, wash 3 times with PBS, 5min each time;

S2.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the regeneration of DRG neuron axons, take pictures and count the distribution of neurite lengths in each group.

Wherein, the specific steps of step S3 are:

S3.1. Extraction of primary DRG neurons

S3.1.1. DRG neurons were obtained from the red skin of SD rats on 1d. The dorsal root ganglia were taken out and placed in dissection solution HA, digested with an appropriate amount of 3mg/ml collagenase, 37°C, 30min;

S3.1.2. Discard collagenase, add an appropriate amount of 0.25% trypsin to digest, 37°C, 20min;

S3.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S3.1.4. Resuspend the cells with neuronal medium, pass through a 200-mesh sieve, and seed them into a 6-well plate coated with polylysine;

S3.2. Transfection of neuronal cells with mimics

S3.3. DRG cell RNA extraction and qRT-PCR

S3.3.1. Collect DRG neuron cells cultured for 3 days in vitro, and extract RNA;

S3.3.2. Use a reverse transcription kit for reverse transcription;

S3.3.3. After reverse transcription, use RT-PCR kit for qRT-PCR;

PCR machine reaction program:

Stage 1: 95℃6min;

Stage 2: 95℃ for 10s, 60℃ for 30s, 72℃ for 10s;

Stage 3: 95℃ for 15s, 60℃ for 1min, 95℃ for 15s;

NRP1 primer sequence:

Forward: CGCCTGAACTACCCTGAA,

Reverse: CCCCACAGCAGTAACGA.

S3.4, Western blot test

S3.4.1. Collect DRG neuron cells cultured for 3 days in vitro, rinse once with PBS, add cell lysate containing 1% protease inhibitor, lyse on ice for 5-10 min, until the cells are completely lysed; centrifuge at 4°C, 13000rpm, 10min, collect supernatant;

S3.4.2, BCA method protein quantification;

S3.4.3. Perform SDS-PAGE electrophoresis, and block with 5% skimmed milk after transfer to the membrane for 2h at room temperature;

S3.4.4. Incubate with primary antibody, dilute anti-NRP1 Polyclonal antibody (1:400) with primary antibody diluent, incubate at room temperature overnight;

S3.4.5, 1×TBS wash 3 times, each time 10min;

S3.4.6. Dilute the secondary antibody goat anti-rabbit HRP with 5% skim milk, incubate at room temperature for 120 min;

S3.4.7, 1×TBST wash 3 times, each time 10min;

S3.4.8, 1×TBS wash once, 10min;

S3.4.9. Incubate the ECL color developing solution on the membrane at room temperature for 1-3 minutes, develop, observe the Western blot results, and transfer the results to the computer with a membrane scanner after the film is dried.

The beneficial effects of the above-mentioned technical solutions of the present invention are as follows:

1. The present invention uses miR-30a-5p as a molecular intervention target, and overexpresses miR-30a-5p to promote the growth and regeneration of DRG neuron axons.

2. The present invention utilizes microfluidics to transfect DRG neurons in vitro with miR-30a-5p mimic, which can significantly promote the growth and regeneration of primary cultured DRG neuron axons.

3. The miR-30a-5p provided by the present invention can participate in the repair of peripheral nerve injury by regulating the growth of DRG neuron axons, which is helpful to better understand the important role of miRNA in the process of nerve injury repair, and is helpful for nerve injury after nerve injury. Therapies offer new targets.

Description of drawings

Fig. 1 is a schematic diagram showing that in vitro overexpression of miR-30a-5p can significantly promote the growth of DRG neuron axons in Example 1 of the present invention;

Figure 2 is a schematic diagram showing that in vitro overexpression of miR-30a-5p can significantly promote the regeneration of DRG neurons axons after injury in Example 2 of the present invention;

Figure 3 is a schematic diagram showing that in vitro overexpression of miR-30a-5p can significantly inhibit the mRNA and protein expression of NRP1 in DRG neurons in Example 3 of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, detailed description will be given below with reference to the accompanying drawings and specific embodiments.

The invention provides a miR-30a-5p, which is used as a molecular target for peripheral nerve injury repair, regulates DRG neurons in the process of peripheral nerve injury repair, and targets Nrp1 to significantly promote the growth and regeneration of DRG neuron axons.

miR-30a-5p is used to prepare drugs for promoting nerve regeneration and repairing nerve injury, wherein the nerve injury is the injury of the sciatic nerve in the peripheral nervous system.

The technical solutions of the present invention are further described below with reference to several specific embodiments.

Example 1: Cultivate primary DRG neuron cells, and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon growth. The specific steps are:

1.1. Microfluidic culture of primary DRG neurons

1.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with an appropriate amount of 3mg/ml collagenase, 37°C, 30min;

1.1.2. Discard the collagenase, add an appropriate amount of 0.25% trypsin for digestion, 37°C, 20min;

1.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

1.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with polylysine. After culturing for 4 h, replace the cells with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine was used to remove non-neuronal cells.

1.2. Transfection of neuronal cells with mimics

After the cells adhered, use

RNAiMAX Reagent was mixed with miR-30a-5p mimics and negative control (Guangzhou Ribo Bio Co., Ltd., the final concentration was 100 nM) and added to the neuron cells cultured in step S1.1. After 8 hours of culture, the medium was replaced with neuron medium.

1.3. Cell immunofluorescence staining and axonal growth length measurement

1.3.1. Discard the cell culture medium 72h after DRG neuron cell transfection, rinse with PBS, add 4% paraformaldehyde, and fix for 30min;

1.3.2. After discarding the paraformaldehyde, wash with PBS three times, 5min each time;

1.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

1.3.4. Dilute the primary antibody anti-Tuj1 antibody (1:400, abcam) with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

1.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

1.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG (1:400, Sigma) with immunohistochemical secondary antibody diluent, add the secondary antibody, and store it at room temperature for 2 hours in the dark;

1.3.7. Discard the secondary antibody, wash 3 times with PBS, 5min each time;

1.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the axonal growth of DRG neurons, take pictures and count the distribution of neurite lengths in each group.

The results show that overexpression of miR-30a-5p (miR-30a-5p mimics) can significantly promote the growth of DRG neuron axons (Fig. 1), wherein, Fig. 1A is Mimic Negative control (Mimic-NC, negative control) or miR-30a-5p mimics were transfected into DRG neurons cultured in microfluidics in vitro, and the cells were immunohistochemically stained 72 h later. The red light is Tuj1/Cy3, and then converted into grayscale image by Photoshop, Bar=200μm. Figure 1B shows the average of at least 15 longest axons in DRG neurons transfected with Mimic-NC and miR-30a-5p mimics in vitro. *P<0.05, ***P<0.001.

Example 2: Cultivate primary DRG neuron cells and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon regeneration. The specific steps are as follows:

2.1. Microfluidic culture of primary DRG neurons

2.1.1. DRG neurons were obtained from the red skin of 1d SD rats. The dorsal root ganglia were taken out and placed in dissection solution HA, digested with an appropriate amount of 3mg/ml collagenase, 37°C, 30min;

2.1.2. Discard the collagenase, add an appropriate amount of 0.25% trypsin for digestion, 37°C, 20min;

2.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

2.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with polylysine. After culturing for 4 hours, replace the cells with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine was used to remove non-neuronal cells.

2.2. Transfection of neuronal cells with mimics

After the cells adhered, use

RNAiMAX Reagent was mixed with miR-30a-5p mimics and negative control (Guangzhou Ribo Bio Co., Ltd., the final concentration was 100 nM) and added to the cultured neurons. After 8 h of culture, the culture medium was replaced with neuron medium.

2.3. Cell immunofluorescence staining and axon regeneration length measurement

2.3.1. After 72 hours of transfection of DRG neuron cells, use negative pressure to remove the axons of the cells in the chamber. After 24 hours, discard the cell culture medium, rinse with PBS, and add 4% paraformaldehyde. Fixed for 30min;

2.3.2. After discarding the paraformaldehyde, wash with PBS three times, 5min each time;

2.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

2.3.4. Dilute the primary antibody anti-Tuj1 antibody (1:400, abcam) with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

2.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

2.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG (1:400, Sigma) with immunohistochemical secondary antibody diluent, add the secondary antibody, and store it at room temperature for 2 hours in the dark;

2.3.7. Discard the secondary antibody and wash 3 times with PBS, 5min each time;

2.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the regeneration of DRG neuron axons, take pictures and count the distribution of neurite lengths in each group.

The results show that overexpression of miR-30a-5p (miR-30a-5p mimics) can significantly promote the regeneration of DRG neuron axons (Fig. 2), wherein, Fig. 2A is Mimic Negative control (Mimic-NC, negative control) or miR-30a-5p mimics were transfected into DRG neurons cultured in microfluidics in vitro, and the growing axons were removed by negative pressure suction after 3 days. Immunohistochemical staining of cells after 24h. The red light is Tuj1/Cy3, and then converted into grayscale image by Photoshop, Bar=100μm. Figure 2B shows the average of at least 15 longest axons in DRG neurons transfected with Mimic-NC and miR-30a-5p mimics in vitro. *P<0.05, ***P<0.001.

Example 3: Extracting primary DRG neuron cells to observe the inhibition of NRP1 mRNA and protein expression by overexpression of miR-30a-5p in vitro. The specific steps are as follows:

3.1. Extraction of primary DRG neurons

3.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with an appropriate amount of 3 mg/ml collagenase, 37 °C, 30 min;

3.1.2. Discard the collagenase, add an appropriate amount of 0.25% trypsin for digestion, 37°C, 20min;

3.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

3.1.4. Resuspend the cells in neuronal medium, and seed them into 6-well plates coated with polylysine after passing through a 200-mesh sieve.

3.2. Transfection of neuronal cells with mimics

After the cells adhered, use

3.3. DRG cell RNA extraction and qRT-PCR

3.3.1. Collect DRG neuron cells cultured for 3 days in vitro, and extract RNA; press

Reagent (Invitrogen) instructions to extract RNA.

3.3.2. Use QIAGEN reverse transcription kit for reverse transcription;

3.3.3. After reverse transcription, use

The PrimeScript RT-PCR Kit (QIAGEN) was used for qRT-PCR; the operation was carried out according to the kit instructions (with GAPDH as the internal reference),

PCR machine reaction program:

Stage 1: 95℃6min;

Stage 2 (Cycle: 40): 95℃ for 10s, 60℃ for 30s, 72℃ for 10s;

Stage 3: 95℃ for 15s, 60℃ for 1min, 95℃ for 15s;

NRP1 primer sequence:

Forward: CGCCTGAACTACCCTGAA,

Reverse: CCCCACAGCAGTAACGA.

The qRT-PCR results are shown in Figure 3A, which showed that overexpression of miR-30a-5p could significantly inhibit the mRNA level of NRP1 in DRG neurons compared with Mimic Negative control group. Among them, Figure 3A shows DRG neurons cultured in vitro transfected with Mimic Negative control (Mimic-NC, negative control) or miR-30a-5p mimics, respectively, and the expression of NRP1 mRNA was detected by qRT-PCR after 72 h, and the internal reference was GAPDH. ***P<0.001. Figure 3B shows DRG neurons cultured in vitro transfected with Mimic Negative control (Mimic-NC, negative control) or miR-30a-5p mimics, respectively. The expression of NRP1 protein was detected by Western blot after 72 h, and the internal control was β-actin. **P<0.01.

3.4. Western blot test

3.4.1. Collect DRG neuron cells cultured for 3 days in vitro, rinse with PBS, add an appropriate amount of cell lysate containing 1% protease inhibitor, and lyse on ice for 5-10 min until the cells are completely lysed; centrifuge at 4°C, 13000rpm, 10min, collect supernatant;

3.4.2. Protein quantification by BCA method;

3.4.3. Perform SDS-PAGE electrophoresis, and block with 5% skimmed milk after transfer to the membrane for 2h at room temperature;

3.4.4. Incubate with primary antibody, dilute anti-NRP1Polyclonal antibody (1:400) with primary antibody diluent, incubate at room temperature overnight;

3.4.5 Wash 3 times with 1×TBS, 10min each time;

3.4.6. Dilute the secondary antibody goat anti-rabbit HRP (1:1000) with 5% skim milk, incubate at room temperature for 120 min;

3.4.7. Wash 3 times with 1×TBST, 10min each time;

3.4.8, 1×TBS wash once, 10min;

3.4.9. Incubate the ECL color developing solution on the membrane, develop at room temperature for 1-3 minutes, observe the results of Western blot, and transfer the results to the computer with a membrane scanner after the film is dried. The Western blot results are shown in Figure 3B, which showed that overexpression of miR-30a-5p could significantly inhibit the protein level of NRP1 in DRG neurons compared with the Mimic Negative control group.

The above are the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims

A miR-30a-5p is characterized in that it is a molecular target of peripheral nerve damage repair.
A use of miR-30a-5p, characterized in that it is used to prepare a drug for promoting nerve regeneration and repairing nerve damage.
The use of miR-30a-5p according to claim 2, wherein the nerve injury is peripheral nervous system sciatic nerve injury.
The application of miR-30a-5p in promoting DRG neuron axon regeneration and peripheral nerve injury repair is characterized in that it includes the following verification steps:

S1. Cultivate primary DRG neuron cells, and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon growth;

S2. Cultivate primary DRG neuron cells, and observe in vitro overexpression of miR-30a-5p to promote DRG neuron axon regeneration;

S3. Extract primary DRG neuron cells to observe the inhibition of NRP1 mRNA and protein expression by overexpression of miR-30a-5p in vitro.
The application of miR-30a-5p in promoting DRG neuron axon regeneration and peripheral nerve injury repair according to claim 4, characterized in that,

The specific steps of step S1 are:

S1.1. Microfluidic culture of primary DRG neuron cells

S1.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with 3mg/ml collagenase, 37°C, 30min;

S1.1.2. Discard collagenase, add 0.25% trypsin to digest, 37℃, 20min;

S1.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S1.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with poly-lysine. After culturing for 4 hours, replace them with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine for depletion of non-neuronal cells;

S1.2. Transfection of neuronal cells with mimics

After the cells adhered, miR-30a-5p mimics and negative control were mixed with transfection reagent, added to the neuron cells cultured in step S1.1, and replaced with neuron medium after 8 hours of culture;

S1.3. Cell immunofluorescence staining and axonal growth length measurement

S1.3.1. Discard the cell culture medium after 72h of DRG neuron transfection, rinse with PBS, add 4% paraformaldehyde, and fix for 30min;

S1.3.2. After discarding the paraformaldehyde, wash with PBS three times for 5 minutes each time;

S1.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

S1.3.4. Dilute the primary antibody anti-Tuj1 antibody with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

S1.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

S1.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG with immunohistochemical secondary antibody diluent, add the secondary antibody, and store it at room temperature in the dark for 2 hours;

S1.3.7. Discard the secondary antibody, wash 3 times with PBS, 5min each time;

S1.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the axonal growth of DRG neurons, take pictures and count the distribution of neurite lengths in each group.
The application of miR-30a-5p in promoting DRG neuron axon regeneration and peripheral nerve injury repair according to claim 4, characterized in that,

The specific steps of step S2 are:

S2.1. Microfluidic culture of primary DRG neuron cells

S2.1.1. DRG neurons were obtained from the red skin of 1d SD rats, and the dorsal root ganglia were taken out and placed in dissection solution HA, digested with 3mg/ml collagenase, 37°C, 30min;

S2.1.2. Discard collagenase, add 0.25% trypsin to digest, 37℃, 20min;

S2.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S2.1.4. Resuspend the cells in DMEM containing 5% FBS, pass through a 200-mesh sieve, and seed them into a microfluidic chamber coated with polylysine. After culturing for 4 hours, replace them with 2% B-27, Neurobasal medium with 2 mM glutamine and 10 ng/ml NGF, 10 mM cytarabine for depletion of non-neuronal cells;

S2.2. Transfection of neuronal cells with mimics

After the cells adhered, the miR-30a-5p mimics and negative control were mixed with transfection reagent and added to the cultured neurons, and the culture medium was replaced after 8 hours of culture;

S2.3. Cell immunofluorescence staining and axon regeneration length measurement

S2.3.1. After 72 hours of transfection of DRG neuron cells, the axons of the cells in the chamber were removed by negative pressure. After 24 hours, the cell culture medium was discarded, rinsed with PBS, and 4% paraformaldehyde was added. Fixed for 30min;

S2.3.2. After discarding the paraformaldehyde, wash with PBS three times for 5 minutes each time;

S2.3.3. Add immunohistochemical blocking solution and block at room temperature for 1 hour;

S2.3.4. Dilute the primary antibody anti-Tuj1 antibody with immunohistochemical primary antibody diluent, add the primary antibody, and store it at 4°C overnight;

S2.3.5. Discard the primary antibody, wash 3 times with PBS, 5min each time;

S2.3.6. Dilute the fluorescent secondary antibody Cy3 sheep anti-mouse IgG with immunohistochemical secondary antibody diluent, add the secondary antibody, and protect from light at room temperature for 2 hours;

S2.3.7. Discard the secondary antibody, wash 3 times with PBS, 5min each time;

S2.3.8. Add an appropriate amount of PBS, observe under a ZEISS upright fluorescence microscope, take pictures, observe the regeneration of DRG neuron axons, take pictures and count the distribution of neurite lengths in each group.
The application of miR-30a-5p in promoting DRG neuron axon regeneration and peripheral nerve injury repair according to claim 4, characterized in that,

The specific steps of step S3 are:

S3.1. Extraction of primary DRG neurons

S3.1.1. DRG neurons were obtained from the red skin of SD rats on 1d. The dorsal root ganglia were taken out and placed in dissection solution HA, digested with an appropriate amount of 3mg/ml collagenase, 37°C, 30min;

S3.1.2. Discard collagenase, add an appropriate amount of 0.25% trypsin to digest, 37°C, 20min;

S3.1.3. Use DMEM containing 10% fetal bovine serum to stop the action of trypsin, and discard the supernatant after centrifugation;

S3.1.4. Resuspend the cells with neuronal medium, pass through a 200-mesh sieve, and seed them into a 6-well plate coated with polylysine;

S3.2. Transfection of neuronal cells with mimics

After the cells adhered, the miR-30a-5p mimics and negative control were mixed with transfection reagent and added to the cultured neurons, and the culture medium was replaced after 8 hours of culture;

S3.3. DRG cell RNA extraction and qRT-PCR

S3.3.1. Collect DRG neuron cells cultured for 3 days in vitro, and extract RNA;

S3.3.2. Use a reverse transcription kit for reverse transcription;

S3.3.3. After reverse transcription, use RT-PCR kit for qRT-PCR;

PCR machine reaction program:

Stage 1: 95℃6min;

Stage 2: 95℃ for 10s, 60℃ for 30s, 72℃ for 10s;

Stage 3: 95℃ for 15s, 60℃ for 1min, 95℃ for 15s;

NRP1 primer sequence:

forward: CGCCTGAACTACCCTGAA,

Reverse: CCCCACAGCAGTAACGA;

S3.4, Western blot test

S3.4.1. Collect DRG neuron cells cultured for 3 days in vitro, rinse once with PBS, add cell lysate containing 1% protease inhibitor, lyse on ice for 5-10 min, until the cells are completely lysed; centrifuge at 4°C, 13000rpm, 10min, collect supernatant;

S3.4.2, BCA method protein quantification;

S3.4.3. Perform SDS-PAGE electrophoresis, and block with 5% skimmed milk after transfer to the membrane for 2h at room temperature;

S3.4.4. Incubate with primary antibody, dilute anti-NRP1 Polyclonal antibody (1:400) with primary antibody diluent, incubate at room temperature overnight;

S3.4.5, 1×TBS wash 3 times, each time 10min;

S3.4.6. Dilute the secondary antibody goat anti-rabbit HRP with 5% skim milk, incubate at room temperature for 120 min;

S3.4.7, 1×TBST wash 3 times, each time 10min;

S3.4.8, 1×TBS wash once, 10min;

S3.4.9. Incubate the ECL color developing solution on the membrane at room temperature for 1-3 minutes, develop, observe the Western blot results, and transfer the results to the computer with a membrane scanner after the film is dried.