Axon regeneration inhibitor and application thereof

Abstract

The invention discloses an axon regeneration inhibitor and application thereof. The axon regeneration inhibitor comprises siNRA and / or shRNA for targeted inhibition of Hic2 gene expression, can be applied to construction of a nerve aplastic disorder model and a nerve repair disorder model, and can also be applied to inhibition of expression of Dynlt1a protein in neuronal cells and regulation of neuronal axon growth. The invention discloses that Hic2 is used as a key molecule for regulating and controlling DRG neuron axon regeneration for the first time, and proposes that Hic2 can influence axon growth by regulating and controlling a dynamic protein Dynlt1a. Compared with the existing research, the invention not only expands the action network of the transcription factor in axon regeneration, but also provides a new intervention target, is expected to provide a theoretical basis and an experimental basis for a treatment strategy of nerve injury repair, and also widens the cognition on the biological function of Hic2.

Description

Technical Field

[0001] The present invention relates to an inhibitor for nerve injury repair research, and in particular to an axon regeneration inhibitor and its application. Background Art

[0002] The repair of nerve injury is an important challenge in clinical medicine. Peripheral nerve injury (PNI) and central nerve injury (CNSI) are common clinical neurological injuries. PNI is mostly caused by trauma, surgery or metabolic diseases, and can lead to paralysis in severe cases. Different from PNI, central nerve injury (CNSI), such as spinal cord injury, often results in irreversible loss of nerve function due to limited regeneration, and has a high incidence and difficult treatment, seriously affecting the quality of life. Improving the intrinsic regenerative ability of neurons is the key to nerve injury repair, and this process depends on the activation and regulation of a series of transcription factors. For example, after PNI, transcription factors such as c-Myc, Sox11, STAT3, Atf3, c-Jun, etc. are rapidly activated, initiating gene expression programs to promote axon growth and regeneration. Existing studies have shown that in mouse retinal ganglion cells, the combination of pluripotent reprogramming transcription factors OSK (Oct4, Sox2, Klf4) can reshape the DNA methylation pattern, promote axon regeneration, and reverse the functional loss caused by aging. Therefore, by targeting the regulation of transcription factors, it may be possible to improve the functional loss caused by aging and promote nerve regeneration, providing a new treatment strategy for nerve injury repair.

[0003] Hic2 (HIC ZBTB transcriptional repressor 2) is a transcriptional regulatory factor that plays an important role in multiple physiological processes, including erythrocyte development, heart development and cancer regulation. Hic2 regulates the fetal-to-adult hemoglobin switch by inhibiting the expression of Bcl11A. Deletion of Hic2 can lead to abnormal heart development. Hic2 transcriptionally activates Sirt1 and has a protective effect in the repair of heart injury. Hic2 also affects the proliferation and invasiveness of tumors in glioblastoma (GBM) by transcriptionally inhibiting the expression of CDK1. So far, no relevant research has been carried out on the specific function of Hic2 in the nerve regeneration system.

[0004] Dynlt1 (Dynein light chain 1), as a component of the cytoplasmic Motor protein dynein complex, plays multiple roles in the process of axon regeneration: 1. Stabilize microtubules to promote the continuous growth of axons; 2. Mediate the retrograde transport of phosphorylated STAT3, transporting injury signals from the injury site to the neuronal cell body in a retrograde manner; 3. Promote the rapid elongation of growth cones by activating Rac1 and actin dynamics.

[0005] Limited axonal regeneration after nerve injury remains a key problem affecting functional recovery. In recent years, the role of transcription factors in axonal regeneration has received attention, but many transcriptional regulatory mechanisms are still unclear. Existing studies have found that transcription factors such as Tgif1 and Sox11 play important roles in axonal growth after peripheral nerve injury. However, there is currently little research on the function of Hic2 in axonal regeneration, and its specific regulatory mechanism is not yet clear. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide an axonal regeneration inhibitor to solve the problem of how to inhibit the axonal regeneration of neuronal cells. Another object of the present invention is to propose the application of an axonal regeneration inhibitor in constructing a nerve regeneration disorder model and a nerve repair disorder model to solve the problem of how to construct a nerve regeneration disorder model and a nerve repair disorder model. The third object of the present invention is to propose the application of an axonal regeneration inhibitor in inhibiting the expression of Dynlt1a protein in neuronal cells to solve the problem of how to inhibit the expression of Dynlt1a protein in neuronal cells.

[0007] Technical Solution: An axonal regeneration inhibitor described in the present invention comprises an RNA inhibitor that targets and inhibits the expression of the Hic2 gene.

[0008] The present invention provides a new intervention target for axonal regeneration with Hic2, and an axonal regeneration intervention strategy centered on Hic2, including but not limited to gene regulation, drug development, RNA interference technology, etc.

[0009] Preferably, the RNA inhibitor includes siNRA and / or shRNA, and the siRNA includes at least one of the following sequences: 5'-GCUUGUAGAUGUGCGUAAAdTdT-3'; 5'-UUUACGCACAUCUACA AGCdTdT-3'; 5'-CCUUUCAAGUGCUCAGUUUdTdT-3'; 5'-AAACUGAGCACUUGAA AGGdTdT-3'; the shRNA includes the following sequence: 5'-GCTGCAACATTTGTGGAAAGATGAAG CTTGATCTTTCCACAAATGTTGCAGC-3'.

[0010] The present invention discloses that the Hic2 gene is a key gene regulating peripheral nerve regeneration. In vitro, inhibiting the expression of Hic2 in DRG neurons using siNRA and / or shRNA can significantly inhibit the axonal regeneration of DRG neurons by inhibiting the expression of the Dynlt1 gene, providing a reliable and convenient way to construct a nerve injury repair disorder model or a nerve regeneration disorder model in vitro.

[0011] The second aspect of the present invention discloses the application of the above axon regeneration inhibitor in constructing a nerve regeneration disorder model and a nerve repair disorder model.

[0012] The present invention first clarifies the function of Hic2 in the nervous system, breaking through the limitation that it is only limited to non-nervous system research, and provides new ideas for its application in nervous system-related research such as nerve injury repair, nerve regeneration, and axon growth regulation, especially its potential application in the field of peripheral nerve injury (PNI) repair. The establishment of the above models can further provide guarantees for the pharmacological and pharmacodynamic studies of nerve injury repair drugs. Based on the above models, it can be observed whether the inhibitory phenomenon of axon regeneration can be reversed or alleviated after drug treatment, and drugs for nerve injury repair treatment can also be designed and synthesized targeting the Hic2 gene target. In addition, the above models can also be used to study the molecular biological mechanisms and signal pathways of axon regeneration disorders.

[0013] Preferably, the method for constructing a nerve regeneration disorder model and a nerve repair disorder model by applying the above axon regeneration inhibitor includes the following steps:

[0014] (1) Isolate and culture primary neuron cells;

[0015] (2) Transfect the axon regeneration inhibitor into the primary neuron cells;

[0016] (3) Digest and resuspend the transfected primary neuron cells, and then re-inoculate and adherently culture the suspension of the transfected primary neuron cells, and observe the axon regeneration situation.

[0017] Preferably, in step (1), the method for isolating and culturing primary neuron cells is: first wash the dorsal root ganglion tissue of the experimental animal with Ca 2 + / Mg2+-free Hank's buffered salt solution, and successively digest the dorsal root ganglion tissue with collagenase and trypsin to obtain a single-cell suspension; filter the single-cell suspension, and resuspend the filtered cells in a medium containing N2 supplement and BSA for conventional incubation.

[0018] Preferably, the transfection method is to co-incubate and culture the liposome or virus loaded with siRNA and / or shRNA with the primary neuron cells.

[0019] The present invention first discovers that Hic2 has the characteristic of co-expression in the nucleus and cytoplasm in DRG neurons, and the gene regulation strategy of Hic2 (including siRNA, shRNA and their delivery vectors AAV, liposome, etc.) can successfully intervene in axon regeneration.

[0020] Preferably, in step (3), the time for adherent culture is 3-7 days.

[0021] Preferably, in step (3), the digestion method is to first digest with 0.02-0.03% trypsin for 10-30 seconds, and then add a complete medium containing 5-15% fetal bovine serum to terminate the digestion reaction.

[0022] The third aspect of the present invention discloses the application of the above axon regeneration inhibitor in inhibiting the expression of Dynlt1a protein in neuronal cells.

[0023] The present invention reveals that Hic2 may regulate axon regeneration by mediating axonal transport through the axon-related motor protein Dynlt1a, and the involved regulatory pathway can be used as a target for intervention.

[0024] The fourth aspect of the present invention discloses the application of the above axon regeneration inhibitor in regulating the growth of neuronal axons.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0026] 1. Reveal the key role of Hic2 in axon regeneration:

[0027] Through RNA-Seq analysis, it was found that Hic2 was significantly up-regulated during axon regeneration, and siRNA-mediated inhibition of Hic2 significantly inhibited the axon growth of DRG neurons. Existing studies mainly focus on transcription factors such as Tgif1 and Sox11, and this study expands the transcriptional regulatory network in nerve injury repair.

[0028] 2. Clarify the possible mechanism of action of Hic2:

[0029] The RNA-Seq results showed that the significant down-regulation of Hic2 significantly reduced the expression of the axon-related motor protein Dynlt1a, suggesting that Hic2 may affect nerve regeneration by regulating the axonal transport system. This discovery provides a basis for exploring new mechanisms of axon growth.

[0030] 3. Advantages compared with the prior art: Current research mainly focuses on the functions of Hic2 in non-neural systems, while the present invention for the first time reveals the role of Hic2 in the nervous system, clarifies its regulatory role in the axon regeneration of peripheral nerve DRG neurons, and expands the understanding of the biological functions of Hic2.

[0031] 4. Provide new targets for nerve injury intervention:

[0032] Currently, there are limited molecular targets for promoting axon regeneration. This study found that Hic2 may be a new key factor regulating axon growth, providing a new research direction for the intervention after nerve injury. In the future, gene therapy strategies can be designed based on Hic2 to optimize the axon regeneration effect and promote clinical application.

[0033] The present invention first reveals that Hic2 is a key molecule regulating axonal regeneration of DRG neurons, and proposes that it may affect axonal growth by regulating the motor protein Dynlt1a. Compared with existing studies, the present invention not only expands the role network of transcription factors in axonal regeneration, but also provides new intervention targets, which is expected to provide a theoretical basis and experimental evidence for the treatment strategy of nerve injury repair, and also broadens the understanding of the biological function of Hic2. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 are the experimental results of the localization and expression of Hic2 in DRG neurons;

[0035] Figure 2 are the experimental results of inhibiting the expression of Hic2 in DRG neurons by in vitro transfection of siRNA-Hic2 and then inhibiting axonal regeneration;

[0036] Figure 3 are the experimental results of inhibiting the expression of Hic2 in DRG neurons by in vitro infection with shRNA-Hic2 AAV and then inhibiting axonal growth;

[0037] Figure 4 are the experimental results of inhibiting the expression of Hic2 in DRG neurons in vitro and then inhibiting the expression of the motor protein (Dynlt1a). DETAILED DESCRIPTION OF THE INVENTION

[0038] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0039] Example 1: Localization and expression of Hic2 in DRG neurons:

[0040] (1) Sciatic nerve crush model and collection of DRG tissues

[0041] Twenty-four 6-week-old male C57BL / 6J mice weighing 18-22 g were randomly divided into 4 groups (untreated, 0 hours, 1 day, 3 days), with 6 mice in each group. An immediate-use anesthetic was administered to the mice at a dose of 0.2 ml / 10 g. The skin of the hind limbs of the mice was disinfected with iodine tincture, and the skin was incised. The muscle was bluntly dissected to expose the sciatic nerve. The sciatic nerve was carefully lifted with microsurgical forceps and clamped with a special mouse nerve clamp for 20 seconds. After clamping, the muscle and skin were sutured, and the suture area was disinfected again with iodine tincture. After the operation, the mice were placed on a heating pad to maintain appropriate temperature and humidity conditions. The mice were subjected to a 12-hour light / dark cycle and had free access to food and water. The dorsal root ganglion (DRG) tissues of the mice were collected at the corresponding time points, placed in cryotubes and stored in liquid nitrogen for subsequent use. The DRG tissues of the untreated group of mice were directly collected and fixed overnight at 4°C with 4% paraformaldehyde for subsequent immunohistochemistry.

[0042] (2) Immunohistochemistry

[0043] DRG tissues fixed overnight at 4°C with 4% paraformaldehyde were embedded in OCT and sectioned using a cryostat with a section thickness of 12 μm. The pre-treated DRG tissues were added with immunohistochemical blocking solution and blocked at room temperature for 1 h. They were incubated with corresponding primary antibodies and specific fluorescent secondary antibodies for labeling gene expression and localization. Tuj1 (1:500, Santa Cruz Biotechnology) antibody was used to label DRG neurons, Hic2 antibody (1:200, abclonal) was used to label Hic2, and DAPI was used to stain cell nuclei. The stained neurons were imaged using a Leica microscope to observe the expression and localization of Hic2 in DRG. The results showed that Hic2 was expressed in both the cytoplasm and cell nuclei of DRG neurons, as shown in Figure 1 Figure A below, where red: Tuj1; green: Hic2; Bar = 50 μm;

[0044] (3) Real-time quantitative PCR (qRT-PCR)

[0045] Total RNA was extracted from DRG tissues collected at different time points (0 h, 1 d, 3 d) using TRIzol reagent from Invitrogen. The extracted RNA was reverse transcribed into cDNA using the Prime-Script RT kit from TaKaRa (Dalian, China) according to the instructions. SYBR Premix Ex Taq reagent from TaKaRa was used for qRT-PCR to identify the expression changes of Hic2 in DRG at different time points after injury. GAPDH was used as an internal reference gene to normalize the expression of the target gene, and the relative expression level was calculated using the 2^-ΔΔCT method. The primers for Hic2 were: Hic2-F CGGCAACACGAGAAGACAC; Hic2-R AACGCATACCACACTCATCAC. The primers for the internal reference were: GAPDH-F ATGCCATCACTGCCACTCA; GAPDH-R CCTGCTTCACCACCTTCTTG. The results showed that the Hic2 mRNA in DRG neurons did not change in the early stage after sciatic nerve crush injury, but began to decrease 3 d after injury, as shown in Figure 1 Figure B below, where all data are Mean±SEM, Students’ t-test, ** represents P<0.01, NS represents no-significant (no statistical significance).

[0046] Example 2: Transfection of siRNA-Hic2 inhibits the expression of Hic2 in DRG neurons and inhibits axonal regeneration

[0047] (1) Isolate and culture primary DRG neurons

[0048] The primary DRG neurons were isolated from the dorsal root ganglia of the L4-L6 segments of normal adult male mice. The DRG tissues were first washed three times with Ca 2 + / Mg2+-free Hank's balanced salt solution (CMF-HBSS), and then digested with 0.3% collagenase at 37 °C for 90 minutes. Subsequently, they were digested with 0.25% trypsin for 25 minutes to obtain a single-cell suspension. The single-cell suspension was filtered through a 70 μm filter and centrifuged to collect the cells. The filtered cells were resuspended in a medium containing N2 supplement and 0.1% BSA, and incubated routinely in a 37 °C incubator for 24 hours, after which the cells were collected for subsequent experimental treatment.

[0049] (2) Transfect DRG neurons with siRNA

[0050] For the siRNA transfection of DRG neurons, Lipofectamine RNAiMAX transfection reagent (Invitrogen) was used and operated according to the instructions. Negative control siRNA (5’-UUCUCCGAACGUGUCACGUdTdT-3’) and Hic2-specific siRNA (Ribobio) were transfected into the cells. The three siRNA-Hic2 double-stranded sequences were as follows:

[0051] siHic2-1 (Hic2 siRNA-1): 5’-GCUUGUAGAUGUGCGUAAAdTdT-3’; 5’-UUUACGCACAUCUACAAGCdTdT-3’.

[0052] siHic2-2 (Hic2 siRNA-2): 5’-GCUAUGAGACAGUGUCAUAdTdT-3’; 5’-UAUGACACUGUCUCAUAGCdTdT-3’.

[0053] siHic2-3 (Hic2 siRNA-3): 5’-CCUUUCAAGUGCUCAGUUUdTdT-3’; 5’-AAACUGAGCACUUGAAAGGdTdT-3’.

[0054] The results showed that siHic2-1 and siHic2-3 in siRNA-Hic2 could significantly knockdown the expression of Hic2 in DRG neurons, as shown in Figure 2 Figure A below, Figure 2Figure A shows the Hic2 mRNA expression levels in DRG neurons transfected with control siRNA or Hic2-specific siRNAs (Hic2 siRNA-1, Hic2 siRNA-2, Hic2 siRNA-3) detected by qRT-PCR to evaluate the knockdown efficiency of each siRNA.

[0055] (3) In vitro resuspension to simulate neuronal injury

[0056] Forty-eight hours after transfection, the DRG cells transfected with siRNA were treated. First, they were digested with 0.025% trypsin for 20 seconds, and then the digestion reaction was terminated by adding complete medium containing 10% fetal bovine serum (FBS). Then, they were centrifuged at 900 rpm for 5 minutes to collect the cell pellet. Finally, the cells were resuspended and re-seeded onto the coverslips of 24-well plates pre-coated with polylysine to simulate neuronal injury. After culturing for 1 day, the re-seeded cells were fixed and subjected to immunocytochemical analysis.

[0057] (4) Cell immunofluorescence staining and measurement of axonal regrowth length

[0058] Seventy-two hours after transfection of DRG neurons, the DRG neurons were first fixed with 4% paraformaldehyde for 30 minutes, then permeabilized with 0.3% Triton X-100 (Sigma) for 10 minutes, and then blocked with immunohistochemical blocking solution (Beyotime) at room temperature for 1 hour. They were incubated with Tuj1 (1:500, Santa Cruz Biotechnology) antibody and specific fluorescent secondary antibody. The stained neurons were imaged using a Leica microscope, photographed, and the distribution of the lengths of the processes in each group was statistically analyzed to evaluate the regeneration of neuronal processes. The results showed that in vitro transfection of siRNA to inhibit the expression of Hic2 significantly inhibited the growth of DRG neuronal processes, as shown in Figure 2 Figures B - D in Figure A. Figure B is a representative immunofluorescence image showing the axonal growth of neurons transfected with control siRNA or Hic2-specific siRNAs (siHic2-1, siHic2-3) after resuspension injury, and Tuj1 was used to label neurons. Scale Bar = 50 μm. Figure C is the result of quantitative statistical analysis of the total neurite length of DRG neurons in Figure B, and Figure D is the result of quantitative statistical analysis of the longest neurite length of DRG neurons in Figure B. Figure 2 All data in Figure A are Mean ± SEM, Students’s t-test, * represents P < 0.05, ** represents P < 0.01, **** represents P < 0.001.

[0059] Example 3: Infection with shRNA-Hic2 inhibits the expression of Hic2 in DRG neurons in vitro and inhibits axonal regeneration

[0060] (1) The steps for separating and culturing primary DRG neurons are the same as those in Example 2.

[0061] (2) Infect DRG neurons with shRNA-Hic2 adeno-associated virus (AAV)

[0062] Use adeno-associated virus (AAV) to infect dorsal root ganglion (DRG) neurons. Directly co-incubate the DRG cells cultured in 6-well plates with negative control AAV or AAV containing shRNA-Hic2, and culture for 7 days at 37 °C. shRNA-Hic2 sequence: 5’-GCTGCAACATTTGTGGAAAGATGAAGCTTGATCTTT CCACAAATGTTGCAGC-3’. The results show that shRNA-Hic2 adeno-associated virus (AAV) can significantly knockdown the expression of Hic2 in DRG neurons, as Figure 3 shown in Figure A of Figure 3 Figure A in is the schematic diagram of the construction of shRNA-Hic2-AAV lentiviral vector and its knockdown efficiency.

[0063] (3) Resuspend in vitro to simulate neuronal injury

[0064] Six days after the infection of adeno-associated virus (AAV), the DRG cells infected with shRNA-Hic2 adeno-associated virus (AAV) are treated. First, digest with 0.025% trypsin for 20 seconds, and then add complete medium containing 10% fetal bovine serum (FBS) to terminate the digestion reaction. Then centrifuge at 900 rpm for 5 minutes to collect the cell pellet. Finally, resuspend the cells and re-seed them on the coverslips of 24-well plates pre-coated with polylysine to simulate neuronal injury. After culturing for 1 day, the re-seeded cells are fixed and subjected to immunocytochemical analysis.

[0065] (4) Cell immunofluorescence staining and measurement of axonal regrowth length

[0066] One week after the infection of DRG neurons with AAV, first fix the DRG neurons with 4% paraformaldehyde for 30 minutes, then permeabilize with 0.3% Triton X-100 (Sigma) for 10 minutes, and then block with immunohistochemical blocking solution (Beyotime) at room temperature for 1 hour. Incubate with Tuj1 (1:500, Santa Cruz Biotechnology) antibody and specific fluorescent secondary antibody. The stained neurons are imaged with a Leica microscope, photographed, and the distribution of the length of the protrusions in each group is statistically analyzed to evaluate the regeneration of neuronal protrusions. The results show that the infection of shRNA-Hic2 adeno-associated virus (AAV) in vitro inhibits the expression of Hic2 and significantly inhibits the growth of DRG neuronal protrusions, asFigure 3 As shown in Figures B - D, in which Figure B shows the axonal growth of neurons infected with the control virus or Hic2 - knockdown virus (Hic2 shRNA) after suspension injury, and Tuj1 was used to label neurons. Scale Bar = 50μm. Figure C shows the results of quantitative statistical analysis of the total neurite length of DRG neurons in Figure B, and Figure D shows the results of quantitative statistical analysis of the longest neurite length of DRG neurons in Figure B. Figure 3 All data in this study are presented as Mean ± SEM, and analyzed by Students’s t - test. *** represents P < 0.001, and **** represents P < 0.0001.

[0067] Example 4: Inhibition of Hic2 expression in DRG neurons in vitro inhibits Dynlt1a expression and Motor protein signaling pathway

[0068] (1) The steps for isolating and culturing primary DRG neurons were the same as those in Example 2.

[0069] (2) siRNA transfection of DRG neurons

[0070] For siRNA transfection of DRG neurons, Lipofectamine RNAiMAX transfection reagent (Invitrogen) was used and operated according to the instructions. Negative control siRNA and Hic2 - specific siRNA (siHic2 - 1) were transfected into the cells. The target sequences of the siRNA - Hic2 double - strand:

[0071] siHic2 - 1: 5’ - GCUUGUAGAUGUGCGUAAAdTdT - 3’; 5’ - UUUACGCACAUCUACA AGCdTdT - 3’.

[0072] (3) RNA - seq and bioinformatics analysis

[0073] 72 hours after transfection with negative control siRNA or Hic2 - specific siRNA, primary DRG neurons were collected. Total RNA of neurons was extracted using Trizol reagent (Sigma) according to the instructions. Subsequently, RNA sequencing was completed by Shanghai OE Biotech Co., Ltd (Shanghai). In bioinformatics analysis, differential expression genes (DEGs) were screened based on fold change, and P - values were calculated by t - test. The screening threshold was set as fold change ≥ 1.5 and P - value ≤ 0.05, and genes with significantly up - regulated or down - regulated expression were considered. The results showed that after knocking down Hic2, 83 genes were up - regulated and 36 genes were down - regulated in primary DRG neurons asFigure 4 As shown in Figure A, the volcano plot shows the significantly upregulated and downregulated genes detected by RNA sequencing in DRG neurons transfected with siRNA-Hic2 (siHic2) compared with the control siRNA (NC). Enrichment analysis of differentially expressed mRNAs was performed using Gene Ontology (GO) data (https: / / geneontology.org / ) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / kegg / ). The results showed that after knocking down Hic2, the genes with differential expression in primary DRG neurons were mainly localized in axons and growth cones. Among them, the downregulated genes were mainly enriched in the motor protein pathway, with Dynlt1a as a typical example. As Figure 4 As shown in Figures B and C, Figure B shows the Gene Ontology Cellular Component analysis of the differentially expressed genes (DEGs) in Hic2-knockdown DRG neurons. The red box indicates that most of the differential genes were significantly enriched in axon- and growth cone-related components; Figure C shows the KEGG pathway analysis of the downregulated genes in Hic2-knockdown DRG neurons. The red box indicates that the downregulated genes were significantly enriched in the motor protein-related pathway.

[0074] (4) Real-time quantitative qRT-PCR

[0075] Total RNA was extracted from primary DRG neurons transfected with negative control siRNA or Hic2-specific siRNA using TRIzol reagent from Invitrogen. The extracted RNA was reverse-transcribed into cDNA using the Prime-Script RT kit from TaKaRa (Dalian, China) according to the instructions. qRT-PCR was used to identify the expression changes of Hic2 in DRG at different time points after injury using SYBR Premix ExTaq reagent from TaKaRa. The expression of the target gene was normalized using GAPDH as an internal reference gene, and the relative expression level was calculated using the 2^-ΔΔCT method. The primers for Dynlt1a were: Dynlt1a-F CCTGCGCAGTCCACTCTAC; Dynlt1a-R ATGGCGCTTTC TATAGCCTCC. The primers for the internal reference were: GAPDH-F ATGCCATCACTGCCACTCA; GAPDH-R CCTGCTTCACCACCTTCTTG. The results showed that knocking down Hic2 in DRG neurons significantly inhibited the expression of the motor protein Dynlt1a, suggesting that Hic2 may regulate axon regeneration by regulating the expression of Dynlt1a. As Figure 4As shown in Figure D, Figure D shows the change in the mRNA expression level of Dynlt1a in DRG neurons with Hic2 knockdown verified by qRT-PCR analysis. Figure 4 All data in this study are Mean±SEM, and the Students’s t-test was used. ** represents P<0.01.

[0076] The present invention identified Hic2 as a key molecule regulating axonal regeneration in DRG neurons. Through in vitro experiments, it was found that Hic2 was expressed in both the nucleus and cytoplasm of DRG neurons, and inhibiting Hic2 (transfecting siRNA-Hic2 or infecting shRNA-Hic2-AAV) significantly inhibited axonal growth. In addition, RNA-Seq data showed that Hic2 might affect axonal regeneration by regulating the axonal-related dynein Dynlt1a, and the expression of Dynlt1a in DRG neurons was significantly reduced after transfecting siRNA-Hic2. This indicates that Hic2 might affect neuronal axonal regeneration by regulating the intracellular axonal transport system.

Claims

1. An axon regeneration inhibitor, characterized in that, An RNA inhibitor that targets and inhibits the expression of the Hic2 gene.

2. The axon regeneration inhibitor according to claim 1, wherein The RNA inhibitor includes siRNA and / or shRNA. The siRNA includes at least one of the following sequences: 5'-GCUUGUAGAUGUGC GUAAAdTdT-3'; 5'-UUUACGCACAUCUACAAGCdTdT-3'; 5'-CCUUUCAAGUGCUC AGUUUdTdT-3'; 5'-AAACUGAGCACUUGAAAGGdTdT-3'. The shRNA includes the following sequence: 5'-GCTGCAACATTTGTGGAAAGATGAAGCTTGATCTTTCCACAAATGTTGCAGC-3'.

3. Use of the axon regeneration inhibitor according to claim 1 or 2 in constructing a nerve regeneration disorder model and a nerve repair disorder model.

4. The application according to claim 3, wherein Comprising the following steps: (1) Isolate and culture primary neuron cells; (2) Transfect the axon regeneration inhibitor into the primary neuron cells; (3) Digest and resuspend the transfected primary neuron cells, and then re-inoculate and adherently culture the suspension of the transfected primary neuron cells, and observe the axon regeneration situation.

5. The application according to claim 4, characterized in that, In step (1), the method for separating and culturing primary neuron cells is as follows: The dorsal root ganglion tissue of the experimental animal is first washed with Ca 2 + / Mg2+-free Hank's buffered salt solution, digested with collagenase and trypsin in sequence, and then a single-cell suspension is obtained; the single-cell suspension is filtered, and the filtered cells are resuspended in a medium containing N2 supplement and BSA and incubated routinely.

6. The application according to claim 4, wherein In step (2), the transfection method is to co-incubate and culture liposomes or viruses loaded with siRNA and / or shRNA with the primary neuron cells.

7. The application according to claim 4, characterized in that, In step (3), the time for adherent culture is 3-7 days.

8. The application according to claim 4, wherein In step (3), the digestion method is to first digest with 0.02-0.03% trypsin for 10-30 seconds, and then add a complete medium containing 5-15% fetal bovine serum to terminate the digestion reaction.

9. Use of the axon regeneration inhibitor according to claim 1 or 2 in inhibiting the expression of Dynlt1a protein in neuron cells.

10. Use of the axon regeneration inhibitor according to claim 1 or 2 in regulating the growth of neuron axons.

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