SiRNA for inhibiting Psmd13 expression and application thereof

Abstract

The invention discloses siRNA for inhibiting expression of Psmd13 and application of the siRNA, and relates to the technical field of gene therapy, the key points of the technical scheme are as follows: by regulating the siRNA expressed by miRNA-29a, the siRNA can specifically target and degrade mRNA of a Psmd13 gene, so that the expression of the Psmd13 is inhibited, the expression of the miR-29a is up-regulated, and the differentiation of neurons is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and more specifically, to siRNA for inhibiting the expression of Psmd13 and application thereof. Background Art

[0002] In recent years, with the aging of the population, the incidence and number of patients with neurodegenerative diseases have also increased significantly, posing huge challenges to society and the medical system. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, amyotrophic lateral sclerosis, etc. In AD, the differentiation and survival of neurons are severely affected, leading to the loss of neurons in the brain. In PD. The loss of dopaminergic neurons is the main pathological feature. Studies have shown that the generation and maintenance of dopaminergic neurons are closely related to the differentiation of stem cells. The differentiation ability of neural precursor cells in patients with Huntington's disease is affected, resulting in reduced neuron generation and increased neuronal death. After a stroke, the differentiation of neural precursor cells and the ability of neuronal regeneration will also be affected. Enhancing the differentiation ability of these cells can help recover from stroke.

[0003] miR-29a is a member of the miR-29 family of microRNAs and plays a vital role in regulating various biological processes, including cell differentiation, proliferation, and apoptosis. miR-29a is highly expressed in the brain and is associated with aging, metabolism, neuronal survival, and neurological diseases. A study found that during postnatal brain development, miR-29a is upregulated in the cortex and hippocampus, associated with neural activity through glutamate receptor activation, directly targeting DCX (doublecortin) to enhance axonal branching, and is essential for neuronal development in mice. More importantly, downregulation of miR-29 a / b1 has been observed in neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. In addition, previous studies have shown that miR-29a levels in the serum of patients with Parkinson's disease are significantly reduced. Mice lacking miR-29 a show signs of premature aging, including weight loss, fat loss, muscle weakness, gait disorders, and increased peristalsis. In rats, knockout of mRNA-29a after MCAO (middle cerebral artery occlusion) led to increased astrocyte proliferation and glutamate release, thereby exacerbating neural damage. Reduced expression of miR-29a may lead to neuroinflammatory responses and neuronal cell death, so it may be a potential therapeutic target for reducing neurodegeneration and protecting cognitive function by regulating miR-29a.

[0004] In summary, miR-29a plays a very important role in the nervous system. Therefore, finding a method to regulate the expression of miR-29a can promote neuronal differentiation without affecting the normal function of miR-29a. At present, the methods for overexpressing miR-29a include plasmid transfection, lentiviral or adenoviral vector expression, chemical synthesis of miR-29a mimics, electroporation, etc. Due to the instability of plasmids, plasmid transfection can only provide short-term overexpression of miR-29a, and the efficiency is low; although lentiviral or adenoviral vectors can achieve long-term miR-29a overexpression with high efficiency, their construction, production and purification processes are complex and expensive, and the use of viral vectors may pose biosafety risks, which will trigger host immune responses in the host and affect the experimental results; chemically synthesized miR-29a mimics are double-stranded structures that simulate natural miRNAs, which can directly bind to target genes in cells to achieve the effect of overexpression. The expression effect of the mimics is short-lived, and high concentrations may have toxic effects on cells. Electroporation can introduce synthetic miR-29a molecules into cells, but it requires specialized equipment and may cause certain damage to cells. At the same time, these methods are all based on DNA overexpression delivery technology, which carries the risk of foreign genes integrating into the genome. Therefore, finding a better method to overexpress miR-29a is currently a technical challenge. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide siRNA for inhibiting the expression of Psmd13 and its application.

[0006] The above technical purpose of the present invention is achieved through the following technical scheme: siRNA that inhibits the expression of Psmd13, the nucleotide sequence of the siRNA positive chain is as shown in SEQ ID NO.1, which is 5'-CCGAUCCUAAUGUGGCUCUUATT-3', and the nucleotide sequence of the siRNA antisense chain is as shown in SEQ ID NO.2, which is 5'-UAAGAGCCACAUUAGGAUCGGTT-3'.

[0007] The application of the siRNA for inhibiting the expression of Psmd13 is that the siRNA sequence is used to prepare a drug for treating neurodegenerative diseases.

[0008] In summary, the present invention has the following beneficial effects: The present invention utilizes the newly discovered upstream gene Psmd13 of miR-29a to knock out its gene, thereby promoting the function of miR-29a. It can be applied to the preparation of products that upregulate the function of miR-29a, and to study the effect of its expression changes on the disease process in disease models related to miR-29a. Compared with the prior art, the present invention regulates the expression of miR-29a by directly knocking out the upstream gene of miR-29a, which can more accurately control the neuronal differentiation process and improve specificity and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a diagram showing the knockdown effect of siRNA molecules on target genes; Figure 2 is the relative expression level of miR-29a after Psmd13 knockout; Figure 3 Representative images of βIII-tubulin-positive cells in undifferentiated mNPCs transfected with siPsmd13 and random siRNA negative control; Figure 4 for Figure 3 Quantitative statistical graph. DETAILED DESCRIPTION

[0010] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0011] 1. Design of siRNA: Sequence design: Search for the mRNA sequence of Psmd13 in GenBank. According to the siRNA design principles, use the siRNA design software of Thermo Fisher Scientific to find a suitable RNAi target sequence. Finally, use BLAST software to perform homology analysis on possible sequences in the GenBank database, and select gene fragments that have high homology with the target gene but no obvious homology with other genes. The designed siRNA is handed over to a biological company for synthesis, and siRNA with a strong silencing effect is selected from the candidate chains for cell experiments.

[0012] The designed siRNR molecules are as follows siRNA1: Sense strand sequence: 5' CCGAUCCUAAUGUGGCUCUUATT 3' (SEQ ID No. 1) Antisense strand sequence: 5' UAAGAGCCACAUUAGGAUCGGTT 3' (SEQ ID No. 2) 2. Neural progenitor cell culture (mNPC) Dissolve PLO (Poly-L-ornithine, PLO) and laminin in DMEM 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 mNPCs into the treated culture dish, add an appropriate amount of DMEM culture medium, and place the culture medium in a 37°C, 5% CO 2 incubator. Every 3-4 days, digest the cells with Accutase. After digestion, transfer the cell suspension to a new treated culture dish and add fresh DMEM culture medium. The NPCs used in the experiment are between passages 2 and 8.

[0013] 3. Transfection of mNPCs (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.

[0014] (2) Prepare transfection complex: Dilute Lipofectamine® RNAiMAX reagent. Add 3µL Lipofectamine® RNAiMAX to 50µL Opti-MEM® medium. Dilute siRNA. Under sterile conditions, add 1µL (10pmol) of siRNA to 50µL Opti-MEM® medium. Add the diluted siRNA to the diluted Lipofectamine® RNAiMAX reagent (ratio 1:1) and incubate at room temperature for 5 minutes.

[0015] (3) Add siRNA-lipid complexes to each well of cell suspension and gently shake to ensure even distribution. Incubate the cells at 37°C for 48 h. Then analyze the transfected cells.

[0016] 4. Analysis of knockdown effect of siRNA molecules on target genes Western blot was used to detect the expression level of Psmd13 gene after siRNA transfection in cells. The specific operation is as follows: After treating the cells with SDS lysis buffer, centrifuge and heat at 100℃ for 10min. After centrifugation, take 5~10 µL of protein sample and load it for SDS-PAGE electrophoresis. Use PVDF membrane for wet transfer. When the transfer is completed, take out the PVDF membrane in time, rinse it with ultrapure water, and put it in the antibody incubation box containing the prepared blocking solution (5% BSA or 5% skim milk). Block it on a shaker at room temperature for 1 hour. Incubate the primary antibody on a shaker at 4℃ overnight. The next day, recover the primary antibody and wash the PVDF membrane with 1× PBST at room temperature for 10 min. Repeat twice. Dilute the corresponding secondary antibody with milk blocking solution at a ratio of 1:20000 and pour it into the incubation box. Incubate it on a shaker at room temperature for 1 hour. Finally, wash the PVDF membrane with 1× PBST for 10 min. Repeat twice. Prepare supersensitive luminescent solution (mix A solution and B solution at a ratio of 1:1), and use a gel imaging scanning system to expose the protein. The results are shown in Figure 1 .

[0017] 5. After siRNA treatment, the expression of miR-29a was detected by RT-qPCR.

[0018] (1) miRNA extraction: RNA was extracted using the miR Vana miRNA extraction kit according to the manufacturer's instructions.

[0019] (2) cDNA synthesis: The extracted miRNA was reverse transcribed into cDNA using the Taqman advanced miRNA cDNA synthesis kit.

[0020] (3) PCR analysis. PCR reactions were performed on a Roche LC480 PCR instrument. Taqman Fast Advance Master Mix was used for detection.

[0021] (4) Normalization of gene expression. The miRNA expression data were normalized using mmu-mir-16 and mmu-mir-191. Figure 2 The results indicated that the relative expression of miR-29a would increase after Psmd13 knockout.

[0022] 6. Neural progenitor cells (mNPCs) differentiation test Quantitative analysis of βIII-tubulin in neural progenitor cells to assess neuronal differentiation (1) Wash the mNPCs transfected with siPsmd13 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.

[0023] (2) Permeabilize with permeabilization buffer (PBS-0.1% Tween20, 5% goat serum) for 30 minutes. Incubate the cells with the specific primary antibody βIII tubulin at 40°C overnight. The next day, wash the cells three times with PBS in the dark for 10 minutes each time.

[0024] (3) Add secondary antibody (Alexa Fluor 546 anti-rabbit) to the cells and incubate for 1 hour at room temperature in the dark. Wash the cells three times with PBS in the dark for 10 minutes each. Counterstain the cells with DAPI for 5 minutes at room temperature in the dark and image them using a confocal SP5 5-channel microscope.

[0025] 7. Image analysis. Analyze images using ImageJ or other image analysis software. Count the number of βIII-tubulin-positive cells in each image and compare with the number of DAPI-stained nuclei. Calculate the percentage of βIII-tubulin-positive cells to assess the degree of neural differentiation. Figure 3 and Figure 4 The results showed that after Psmd13 was knocked out, the number of βIII-tubulin-positive cells increased.

[0026] 8. Statistical analysis: GraphPad Prism 8 was used for statistical analysis. Data points were expressed as Mean ± SEM. Two-tailed t-test was used to calculate P values. P < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01.

[0027] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A siRNA for inhibiting the expression of Psmd13, characterized in that: The nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO. 1, which is 5'-CCGAUCCUAAUGUGGCUCUUATT-3', and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO. 2, which is 5'-UAAGAGCCACAUUAGGAUCGGTT-3'.

2. The use of siRNA for inhibiting Psmd13 expression according to claim 1, characterized in that: The siRNA sequence is used for preparing medicine for treating neurodegenerative diseases.

Images