Use of miR-345-3p inhibitor in preparation of a product for promoting differentiation of neural stem cells in vitro
By using miR-345-3p inhibitors to downregulate miR-345-3p expression in neural stem cells, their differentiation into neurons is promoted, solving the problem of insufficient differentiation of neural stem cells in existing technologies and improving the treatment effect of nervous system diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANTONG UNIV
- Filing Date
- 2026-03-16
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168540A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of miR-345-3p inhibitor in the preparation of products that promote the differentiation of neural stem cells in vitro. Background Technology
[0002] Neural stem cells (NSCs) are a type of cell with self-renewal capacity and multi-lineage differentiation potential. They can differentiate into various nerve cell types, such as neurons, astrocytes, and oligodendrocytes, and play a crucial role in nervous system development, tissue repair, and nerve regeneration. In recent years, with the deepening research on nervous system diseases, the differentiation regulation mechanisms of neural stem cells have gradually become a research hotspot in the field of neuroscience.
[0003] During neural stem cell differentiation, microRNAs (miRNAs), as endogenous small non-coding RNAs, regulate gene expression at the posttranscriptional level by binding to the 3' untranslated region (3'UTR) of target mRNAs, inhibiting their translation or promoting their degradation. The differentiation process of neural stem cells is finely regulated by multiple factors, including extracellular signaling molecules, intracellular signaling pathways, and gene expression networks. In neurological diseases, the differentiation capacity of neural stem cells is often inhibited, leading to a decline in the repair and regeneration capacity of neural tissue. Therefore, finding drugs that can effectively promote neural stem cell differentiation has significant clinical implications.
[0004] In recent years, with in-depth research on the role of miRNAs in the regulation of neural stem cell differentiation, miRNAs, as potential drugs to promote neural stem cell differentiation, have gradually attracted attention. miRNAs regulate the gene expression network within neural stem cells by specifically binding to their target genes, thereby promoting the differentiation of neural stem cells into neural cell types such as neurons.
[0005] In summary, miRNAs show great promise in the preparation of drugs that promote neural stem cell differentiation. This invention aims to provide a pharmaceutical composition based on miR-345-3p inhibitors to promote neural stem cell differentiation, thereby offering new strategies and methods for the treatment of nervous system diseases. Summary of the Invention
[0006] The purpose of this invention is to provide the application of miR-345-3p inhibitor in the preparation of products that promote the differentiation of neural stem cells into neurons in vitro.
[0007] The National Center for Biotechnology Information (NCBI) in the United States created and maintains the public gene chip database GEO (GeneExpression Omnibus), which contains high-throughput gene expression data submitted by numerous research institutions worldwide. For example... Figure 1 As shown, this invention utilizes the GEO database with "neuronal differentiation" and "microRNA" as keywords to retrieve the dataset GSE32122, "MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation." GEO2R analysis identified differentially expressed miRNAs during neuronal differentiation, among which miR-345-3p was one of the significantly underexpressed miRNAs. This suggests that low expression of miR-345-3p may promote the differentiation of neural stem cells into neurons. Therefore, substances that promote low expression of miR-345-3p could serve as drugs to promote the differentiation of neural stem cells into neurons.
[0008] This invention uses miRNA inhibitors to downregulate miR-345-3p expression, with miR-NC as a control. Neural stem cells containing both miR-345-3p inhibitors and control miR-NC were transfected with Lipofectamine 2000 transfection reagent and then cultured in vitro for differentiation. Neuronal differentiation was observed. Results showed that miR-345-3p inhibitors can promote the differentiation of neural stem cells into neurons in vitro. P <0.05%, low expression of miR-345-3p can significantly promote the differentiation of neural stem cells into neurons ( P <0.05). Therefore, substances that promote low expression of miR-345-3p can be used in the preparation of products that promote the differentiation of neural stem cells into neurons in vitro. Attached Figure Description
[0009] Figure 1 Using dataset GSE32122 from the public gene chip database GEO, GEO2R analysis was used to identify differentially expressed miRNAs during neuronal differentiation, among which miR-345-3p was one of the significantly underexpressed miRNAs.
[0010] Figure 2The results are shown in the DCX immunofluorescence assay after differentiation and culture. A represents the DCX immunofluorescence assay results after 48 hours of differentiation and culture of neural stem cells transfected with miR-NC or miR-345-3p inhibitor, with Hoechst nuclear staining and a scale bar of 25 micrometers. B represents the statistical proportion of DCX-positive neuronal precursor cells (mean ± standard deviation).
[0011] Figure 3 The results are as follows: A shows the MAP-2 immunofluorescence assay results after differentiation and culture of neural stem cells transfected with miR-NC or miR-345-3p inhibitor for 7 days. Hoechst staining was used for the nuclei, and the scale bar was 25 micrometers. B shows the statistical results of the proportion of MAP-2 positive mature neurons (mean ± standard deviation). Detailed Implementation
[0012] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0014] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1
[0015] The in vitro neural stem cell differentiation experiment into neurons in this embodiment used rat embryonic neural stem cells.
[0016] In this embodiment, miRNA inhibitors were used to downregulate the expression of miR-345-3p, with miR-NC as a control. Neural stem cells were screened by transfecting the miR-345-3p inhibitor and the control miR-NC with Lipofectamine 2000 transfection reagent to obtain neural stem cells with stable low expression of miR-345-3p and neural stem cells with normal expression of miR-345-3p. Then, they were differentiated and cultured in vitro to observe the differentiation of neurons.
[0017] The specific process is as follows: 1. Neural stem cell culture and miRNA inhibitor transfection (1) The neural stem cell line was Rat Fetal Neural Stem Cells from Gibco, Thermo Fisher Scientific. The cells were cultured in a 37 ℃, 5% CO2 incubator in DMEM / F12 (Gibco), which contained epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at a final concentration of 20 ng / mL, as well as 1% penicillin / streptomycin solution.
[0018] (2) miR-345-3p inhibitor and control miR-NC were purchased from Thermo Fisher Scientific. Cell transfection was performed using Invitrogen's Lipofectamine 2000 transfection reagent, strictly following the reagent instructions. The transfection concentration of the miRNA inhibitor was set at 5 nM.
[0019] 2. Differentiation culture and DCX and MAP-2 immunofluorescence detection: (1) Differentiation culture experiment: Neural stem cells and neural stem cells transfected with miRNA inhibitors / control miR-NC were cultured at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of [number] cells / mL into each well of a 24-well culture plate pre-placed with poly-L-lysine-coated coverslips, and 2 mL of DMEM / F12 complete culture medium containing 10% fetal bovine serum (FBS) was added to each well. The culture plates were then incubated in a humidified incubator at 37 °C and 5% CO2. After 48 hours of culture, cells were detected by double corticosteroid (DCX) immunofluorescence; after 7 days of culture, microtubule-associated protein 2 (MAP-2) immunofluorescence was performed.
[0020] (2) DCX Immunofluorescence Detection: After 48 hours of cell culture, 6-well cell samples were taken from each group. First, the culture medium in the wells was aspirated, and then 1 mL of 0.01 mol / L PBS (pH 7.2) containing 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 minutes. After fixation, the paraformaldehyde solution was aspirated, and the cells were washed 3 times with 0.01 mol / L PBS (pH 7.2). Next, 200 µL of 0.01 mol / L PBS (pH 7.2) containing 10% goat serum was added to each well, and the cells were gently shaken at room temperature for 30 minutes to block non-specific binding sites. After that, the blocking solution was aspirated, and 200 µL of guinea pig anti-DCX antibody (Abcam) diluted to 1:1000 with 0.01 mol / L PBS (pH 7.2) was added, and the cells were gently shaken at room temperature for 1 hour. Then, the culture plates were incubated overnight at 4 °C. The following morning, the primary antibody solution was aspirated, and the cells were washed three times with PBS. Then, 200 µL of Alexa Fluor® 488 goat anti-guinea pig secondary antibody (Abcam) diluted 1:800 with 0.01 mol / L PBS (pH 7.2) was added, and the cells were gently shaken at room temperature for 2 hours in the dark. After secondary antibody incubation, the cells were washed three times with PBS, followed by the addition of 200 µL of Hoechst dye (Abcam) diluted 1:2000 with 0.01 mol / L PBS (pH 7.2), and incubated at room temperature in the dark for 30 minutes. Finally, the cells were thoroughly washed with PBS, mounted with glycerol phosphate buffer, and the expression of DCX immunofluorescence was observed under a fluorescence microscope.
[0021] (3) MAP-2 Immunofluorescence Detection: After 7 days of cell culture, 6-well cell samples were taken from each group. First, the culture medium in the wells was aspirated, and then 1 mL of 0.01 mol / L PBS (pH 7.2) containing 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 minutes. After fixation, the paraformaldehyde solution was aspirated, and the cells were washed 3 times with 0.01 mol / L PBS (pH 7.2). Next, 200 µL of 0.01 mol / L PBS (pH 7.2) containing 10% goat serum was added to each well, and the cells were gently shaken at room temperature for 30 minutes to block non-specific binding sites. After that, the blocking solution was aspirated, and 200 µL of rabbit anti-MAP-2 antibody (Abcam) diluted to 1:1000 with 0.01 mol / L PBS (pH 7.2) was added, and the cells were gently shaken at room temperature for 1 hour. Then, the culture plates were incubated overnight at 4 °C. The following morning, the primary antibody solution was aspirated, and the cells were washed three times with PBS. Then, 200 µL of Alexa Fluor® 488 goat anti-rabbit secondary antibody (Abcam) diluted 1:800 with 0.01 mol / L PBS (pH 7.2) was added, and the cells were gently shaken at room temperature for 2 hours in the dark. After secondary antibody incubation, the cells were washed three times with PBS, followed by the addition of 200 µL of Hoechst dye (Abcam) diluted 1:2000 with 0.01 mol / L PBS (pH 7.2), and incubated at room temperature in the dark for 30 minutes. Finally, the cells were thoroughly washed with PBS, mounted with glycerol phosphate buffer, and the expression of MAP-2 immunofluorescence was observed under a fluorescence microscope.
[0022] 3. Statistical processing SPSS 21.0 statistical software was used to analyze the data. The obtained measurement data conformed to a normal distribution according to the Kolmogorov-Smirnova test and were expressed as mean ± standard deviation. One-way ANOVA was used for pairwise comparisons among the three groups. P A value <0.05 is considered statistically significant.
[0023] The results are as follows Figure 2 , 3 As shown, compared with the blank control group and the miR-NC group, the number of neural stem cells transfected with miR-345-3p inhibitor differentiating into bicorticotinic acid (DCX) positive neuronal precursor cells and microtubule-associated protein 2 (MAP-2) positive mature neurons was significantly increased. One-way ANOVA results showed that the differences among the groups were statistically significant. P <0.05).
Claims
1. Application of miR-345-3p as a target in screening products that promote the differentiation of neural stem cells into neurons in vitro.
2. The application according to claim 1, characterized in that, By downregulating miR-345-3p expression, neural stem cells are promoted to differentiate into neurons.
3. Application of miR-345-3p inhibitor in the preparation of products that promote the differentiation of neural stem cells into neurons in vitro.
4. The application according to claim 3, characterized in that, The miR-345-3p inhibitor is selected from one or more of small molecule compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides, or nucleic acids.
5. A method for promoting the differentiation of neural stem cells into neurons in vitro, characterized in that, Neural stem cells were cultured in a culture medium, and then transfected with miR-345-3p inhibitor. The transfected neural stem cells were then cultured for differentiation to obtain neurons.
6. The method according to claim 5, characterized in that, The culture medium is DMEM / F12 medium, and the DMEM / F12 medium contains epidermal growth factor and basic fibroblast growth factor at a final concentration of 20 ng / mL, as well as 1% penicillin / streptomycin solution.
7. The method according to claim 5, characterized in that, The culture medium used for the differentiation culture was DMEM / F12 complete culture medium containing 10% fetal bovine serum.