Method for promoting neuron regeneration and siRNA

Abstract

The invention relates to a method for promoting neuron regeneration, which is characterized in that miR-9 is overexpressed by silencing an upstream regulatory gene Pannexin2 of miR-9, so that neuronal cells are promoted to differentiate and regenerate. The siRNA for silencing the miR-9 is as shown in SEQ ID NO: 1. According to the invention, the expression of the miR-9 is regulated and controlled by directly knocking out the upstream gene Pannexin2 of the miR-9, so that the neuronal differentiation process can be controlled more accurately, and the specificity and effectiveness are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and specifically to an upstream gene Panx2 that regulates microRNA-9, and siRNA used to inhibit the expression of Panx2, thereby promoting the expression of microRNA-9 and promoting neuron regeneration. Background Art

[0002] In recent years, with the aging of the population, the incidence and number of patients with neurodegenerative diseases have increased significantly, posing a huge challenge to society and the medical system. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and amyotrophic lateral sclerosis.

[0003] MicroRNA (miRNA, miR) is essential for many cellular processes (including neurogenesis), and has two mature chains, miR-9-3p and miR-9-5p. The main chain miR-9-5p is usually called miR-9. In recent years, researchers have discovered and confirmed that miR-9 is involved in the development and differentiation of stem cells and neurons, as well as the maintenance of traits, and plays an indispensable role in stem cells and neurons. Finding a method to overexpress miR-9 can promote neuronal differentiation without affecting the normal function of miR-9. Currently, there are plasmid transfection, lentiviral or adenoviral vector expression, and chemically synthesized miR-9 mimics. Plasmid transfection can only provide short-term overexpression of miR-9 and has low efficiency. The construction, production and purification process of lentiviral or adenoviral vectors is complex and expensive, and the use of viral vectors may pose a biosafety risk, which will induce host immune responses in the host and affect the experimental results. The expression effect of chemically synthesized miR-9 mimics is short-lived, and high concentrations may have toxic effects on cells. Chemically synthesized miR-9 mimics are expensive. At the same time, these methods are all based on DNA overexpression delivery technology, which has the risk of foreign genes integrating into the genome. Therefore, finding a better method to overexpress miR-9 is currently a technical challenge.

[0004] The Pannexin2 gene is a member of the ubiquitin protein family. Previous studies have shown that blocking the expression of Pannexin2 can inhibit the proliferation, migration and invasion of prostate cancer cells. No studies have shown any connection between the Panx2 gene and the expression of miR-9.

[0005] RNA interference (RNAi) refers to a gene silencing phenomenon induced by double-stranded RNA in molecular biology. Its mechanism is to inhibit gene expression by hindering the transcription and translation of specific genes. This technology has specificity and high efficiency, simple method and short cycle, and is increasingly gaining attention.

[0006] Small interfering RNA (siRNA) can regulate gene expression through RNAi. siRNA is usually a 21-nucleotide double-stranded RNA (dsRNA). siRNA can be cut into longer double-stranded RNA or small hairpin RNA by dicer enzyme; siRNA can also be introduced into cells through a variety of different transfection techniques and produce a specific gene knockout effect on a specific gene.

[0007] In summary, siRNA provides a way to silence upstream regulatory genes and thus overexpress miR-9 to achieve the purpose of promoting neuronal regeneration. Summary of the invention

[0008] The purpose of the present application is to provide a method for overexpressing miR-9 by silencing upstream genes, so as to solve the problem of promoting increased neuronal differentiation and delaying the onset of neurodegenerative diseases.

[0009] To achieve the above object, the present invention provides a method for promoting neuron regeneration, by silencing Pannexin2, an upstream regulatory gene of miR-9, to overexpress miR-9, thereby promoting the differentiation and regeneration of neuronal cells.

[0010] The siRNA for silencing the Pannexin2 gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0011] A siRNA, the siRNA having a nucleotide sequence as shown in SEQ ID NO: 1, is used in a drug or preparation capable of inhibiting or delaying neurodegenerative diseases.

[0012] Furthermore, siRNA is used in drugs or preparations that promote the expression of miR-9.

[0013] Furthermore, siRNA was applied to disease models related to miR-9 to study the effects of its expression changes on the progression of neurodegenerative diseases.

[0014] The beneficial effects of the present invention are as follows: the present invention regulates the expression of miR-9 by directly knocking out the upstream gene Panx2 of miR-9, which can more accurately control the neuronal differentiation process and improve specificity and effectiveness. The regulatory effect of the Panx2 gene on the expression of miR-9 and the effect of promoting neuronal differentiation are discovered for the first time, which is highly innovative. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the relative expression level of miR-9 after siRNA silencing.

[0016] Figure 2 Representative images of βIII-tubulin-positive cells in differentiated mNPCs (A) and the percentage of βIII-tubulin-positive cells (B). DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0018] Example 1. Design of siRNA

[0019] Sequence design: The mRNA sequence of Pannexin2 was searched in GenBank. According to the siRNA design principles, the siRNA design software of Thermo Fisher Scientific was used to find a suitable RNAi target sequence. Finally, the BLAST software was used to perform homology analysis on possible sequences in the GenBank database, and gene fragments with high homology to the target gene but no obvious homology to other genes were selected. The siRNA designed by the biological company was synthesized, and the siRNA with stronger silencing effect was selected from the candidate chains for cell experiments. The obtained siRNA sense chain sequence is shown in SEQ ID NO: 1. The antisense chain sequence is shown in SEQ ID NO: 2.

[0020] Example 2: Neural progenitor cell culture

[0021] Dissolve poly-L-ornithine (PLO) and laminin in DMEM medium and prepare according to the manufacturer's instructions. Add the prepared solution to the culture dish to ensure that the entire surface is covered. Incubate for a period of time, remove the excess solution and wash the culture dish with sterile PBS. Inoculate neural progenitor cells (mNPCs) into the treated culture dish, add an appropriate amount of DMEM culture medium, and culture in a 37°C, 5% CO2 incubator. Digest the cells with cell digestion solution (Accutase) every 3-4 days. After digestion, transfer the cell suspension to a new treated culture dish and add fresh DMEM culture medium.

[0022] Example 3: Transfection of neural progenitor cells

[0023] 1. Prepare cells: Use Accutase to separate mNPCs from the culture plate. Centrifuge at 200 rpm for 5 minutes at room temperature, remove the supernatant, and retain the cell pellet. Resuspend the cell pellet in complete growth medium and distribute the resuspended cells into the pre-coated 24-well plate, adding an appropriate amount of cell suspension to each well.

[0024] 2. Prepare transfection complexes:

[0025] (1) Dilution RNAiMAX reagent. 3 μL Add RNAiMAX to 50 μL in culture medium.

[0026] (2) Dilute siRNA. Under sterile conditions, add 1 μL (10 pmol) of siRNA to 50 μL in culture medium.

[0027] (3) Add the diluted siRNA to the diluted RNAiMAX reagent (ratio 1:1) and incubate at room temperature for 5 minutes.

[0028] 3. Add siRNA-lipid complexes to each well of cell suspension and gently shake to ensure even distribution. Incubate cells at 37°C for 48h. Then analyze the transfected cells.

[0029] Example 4: Analysis of the silencing effect of siRNA molecules on target genes

[0030] The expression of miR-9 was detected by RT-PCR.

[0031] 1. miRNA extraction: RNA was extracted using the miR Vana miRNA extraction kit according to the manufacturer's instructions.

[0032] 2. cDNA synthesis: Use Taqman advanced miRNA cDNA synthesis kit to reverse transcribe the extracted miRNA into cDNA.

[0033] 3. PCR analysis: PCR reaction was performed on a Roche LC480 PCR instrument. Taqman Fast Advance Master Mix was used for miRNA detection.

[0034] 4. Standardize gene expression. The miRNA expression data were standardized using mmu-mir-16 and mmu-mir-191. The results are shown in Figure 1 , where siPanx2 is the relative expression of miR-9 after interference silencing Panx2, and siControl is a random siRNA negative control. The results show that the relative expression of miR-9 will increase after knocking out Pannexin2, indicating that the Panx2 gene has an upstream regulatory inhibitory effect on miR-9.

[0035] Example 5: Neural progenitor cell differentiation test

[0036] The degree of neuronal differentiation was assessed by quantitative analysis of βIII-tubulin in neural progenitor cells.

[0037] 1. Culture mNPCs transfected with siPanx2 on microscope slides in growth medium at 37°C in a 5% CO2 incubator overnight.

[0038] 2. Wash the cells twice with PBS to remove the culture medium. Fix the cells with 4% paraformaldehyde and incubate at room temperature for 10-20 minutes. Wash the cells again with PBS three times, 5 minutes each time.

[0039] 3. Treat the cells with 0.1-0.5% Triton X-100 permeabilization solution and incubate at room temperature for 10 minutes. Wash the cells three times with PBS.

[0040] 4. Antibody staining. Block cells with 5% BSA in PBS and incubate at room temperature for 1 hour. Incubate cells with βIII-tubulin antibody at 4°C overnight. Wash cells three times with PBS. Incubate cells with Alexa Fluor 488 anti-rabbit secondary antibody at room temperature for 1 hour in the dark. Wash cells three times with PBS.

[0041] 5. Nuclear staining: Use DAPI staining solution to stain the cell nucleus and incubate at room temperature for 5 minutes. Wash the cells three times with PBS.

[0042] 6. Microscope observation: Use 3i Marianas microscope system to observe and take fluorescence images.

[0043] 7. Image analysis. Use image analysis software such as ImageJ to analyze images. Count the number of βIII-tubulin-positive cells in each image and compare it with the number of cell nuclei stained with DAPI. Calculate the percentage of βIII-tubulin-positive cells to assess the degree of neural differentiation. Results are shown in Figure 2 The results showed that after silencing Pannexin2, the number of βIII-tubulin-positive cells increased, indicating that neuronal differentiation increased after knocking out Pannexin2.

[0044] It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

Claims

1. A method for promoting neuron regeneration, characterized in that: By silencing Pannexin2, the upstream regulatory gene of miR-9, miR-9 is overexpressed, thereby promoting the differentiation and regeneration of neuronal cells.

2. The method for promoting neuron regeneration according to claim 1, characterized in that: The siRNA for silencing the Pannexin2 gene has a nucleotide sequence as shown in SEQ ID NO:

1.

3. A siRNA, characterized in that: The siRNA with a nucleotide sequence as shown in SEQ ID NO: 1 is used in drugs or preparations that can inhibit or delay neurodegenerative diseases.

4. The siRNA according to claim 3, characterized in that The siRNA is used in a drug or preparation for promoting the expression of miR-9.

5. The siRNA according to claim 3, characterized in that The siRNA is applied to a disease model associated with miR-9 to study the effect of its expression changes on the progression of neurodegenerative diseases.

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