Application of miR-29a-5p and analogue thereof

Abstract

The invention discloses application of miR-29a-5p and analogues thereof, and particularly relates to application of miR-29a-5p and analogues thereof in preparation of drugs for promoting directional differentiation of neural stem cells. A research result shows that the miR-29a-5p and the analogue thereof can inhibit proliferation of neural stem cells (P is less than 0.05) in vitro and promote the neural stem cells to be differentiated into neurons (P is less than 0.05). According to the experimental results, a basis can be provided for the application of the miR-29a-5p and the analogue thereof to the preparation of the medicine for promoting the directional differentiation of the neural stem cells into the neurons, a new method is provided for the directional differentiation of the neural stem cells into the neurons, and the miR-29a-5p and the analogue thereof are expected to become effective medicine components for treating many neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of miR-29a-5p and its analogs. Background Art

[0002] Neural stem cells (NSCs) are a type of multipotent stem cell that can self-renew and differentiate into multiple types of cells, including neurons, astrocytes, and oligodendrocytes. NSCs are mainly found in the developing central nervous system of mammals, but in adulthood, some NSCs are still retained in local areas of the central nervous system. Neuronal loss is a pathological feature of many neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.). Since NSCs have the potential to differentiate into neurons, NSCs therapy can be used as a promising treatment method. However, the proportion of NSCs that differentiate into neurons in the adult brain is very low, far from enough to completely replenish the lost neurons, so it is necessary to find a way to promote the differentiation of NSCs into neurons.

[0003] MicroRNA (miRNA) is a short non-coding RNA with a length of about 20 to 24 nucleotides. It can regulate the post-transcriptional expression of target genes by blocking the translation of target gene mRNA or accelerating its degradation. miRNAs play an important role in cell proliferation, differentiation, migration and apoptosis by regulating the expression of their target genes. miRNAs are abundant in the central nervous system and play a key role in neurogenesis and neurophysiological processes. They can also play an important role in stem cell fate determination by controlling the expression of many stem cell regulatory factors.

[0004] Based on this, if miRNAs that can promote the differentiation of neural stem cells into neurons are found, it may provide a new method for the directed differentiation of neural stem cells into neurons. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the application of miR-29a-5p and its analogs. The present invention finds through experiments that miR-29a-5p and its analogs can promote the differentiation of neural stem cells into neurons, providing a new method for the directed differentiation of neural stem cells into neurons.

[0006] The present invention is achieved through the following technical solutions:

[0007] Application of miR-29a-5p and its analogs in the preparation of drugs for promoting the directional differentiation of neural stem cells.

[0008] Preferably, the nucleotide sequence of miR-29a-5p is as shown in SEQ ID NO.1.

[0009] Use of the above-mentioned miR-29a-5p and its analogs in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

[0010] Preferably, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0011] Preferably, the miR-29a-5p and its analogs prevent and / or treat neurodegenerative diseases by promoting the directional differentiation of neural stem cells into neurons.

[0012] A pharmaceutical composition for treating neurodegenerative diseases, comprising the above-mentioned miR-29a-5p and its analogs, and pharmaceutically acceptable excipients.

[0013] Preferably, the auxiliary materials include one or more of diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants and adsorption carriers.

[0014] Preferably, the pharmaceutical composition is a non-oral dosage form.

[0015] Preferably, the pharmaceutical composition is a tablet, capsule, powder, pill, granule, solution, suspension, injection, suppository or spray.

[0016] The beneficial effects of the present invention are as follows:

[0017] The research results of the present invention show that miR-29a-5p and its analogs can inhibit the proliferation of neural stem cells in vitro (P < 0.05) and promote the differentiation of neural stem cells into neurons (P < 0.05). The above experimental results can provide a basis for the use of miR-29a-5p and its analogs in the preparation of drugs that promote the directional differentiation of neural stem cells into neurons, and provide a new method for the directional differentiation of neural stem cells into neurons. MiR-29a-5p and its analogs are expected to become effective drug ingredients for the treatment of many neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Results of cell proliferation detection experiments after primary neural stem cells were transfected with miR-NC or miR-29a-5p analogs: A is the expression of cell proliferation marker molecules Cdk2, Ccnd1 and Pcna detected by real-time quantitative PCR; B is the cell growth curve drawn by MTS method at 0, 1, 2, 3 and 4 days after transfection; C is the flow cytometry detection of cell cycle and the statistics of the proportion of cells in each phase; D is the statistics of EdU incorporation experiment and the proportion of EdU positive cells, Hoechst stained nucleus, scale = 200μm; data are expressed as mean ± standard error, * P<0.05, ** P<0.01, *** P < 0.001;

[0019] Figure 2 Results of cell differentiation detection experiments after primary neural stem cells were transfected with miR-NC or miR-29a-5p analogs: A is the expression of neuronal marker molecules Map2, Syn1, Neurod1 and Stmn2 and astrocyte marker molecule Gfap detected by real-time quantitative PCR; B is the expression of Map2 and Gfap detected by protein immunoblotting and the relative gray value of the bands was counted, with β-actin as the internal reference; C is the expression of neuronal marker molecule Tuj1 and astrocyte marker molecule Gfap detected by immunofluorescence, and the proportion of Tuj1-positive cells was counted, Hoechst stained nuclei, scale = 200 μm; D is the flow cytometry detection and the proportion of Tuj1-positive cells was counted; data are expressed as mean ± standard error, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The experimental animals used in the following examples are SD rats from the Experimental Animal Center of Nantong University.

[0024] Example 1

[0025] 1. Isolation, culture and differentiation system of primary neural stem cells

[0026] (1) Isolation and culture of primary neural stem cells

[0027] The hippocampal tissue of embryonic SD rats at E14.5 days was obtained, and after repeated blowing and centrifugation, it was resuspended in neural stem cell culture medium (DMEM / F12+B27+EGF+bFGF+double antibody), filtered with a 40μm filter and seeded into a culture bottle, marked as P0, and passaged after 5-7 days. It was digested with 0.25% trypsin at 37°C for 4-5 minutes, and 5-7mL of stop solution (DMEM / F12+10% fetal bovine serum+double antibody) was added. After repeated blowing and centrifugation, it was resuspended in neural stem cell culture medium and seeded into a culture bottle, marked as P1. After 5-7 days of culture, it was passaged and subsequent experiments were performed.

[0028] (2) Differentiation system

[0029] The P1 neural stem cells were digested with 0.25% trypsin at 37°C for 4-5 min, 5-7 mL of stop solution (DMEM / F12+10% fetal bovine serum+double antibody) was added, and after repeated pipetting and centrifugation, they were resuspended with neural stem cell differentiation solution (DMEM / F12+B27+10% fetal bovine serum+double antibody), filtered with a 40 μm filter and then seeded with cells.

[0030] 2. Transfection of miRNA analogs

[0031] The nucleotide sequence of miR-29a-5p is as follows:

[0032] miR-29a-5p (SEQ ID NO. 1): 5'-ACUGAUUUCUUUUGGUGUUCAG-3'.

[0033] This example uses miR-29a-5p analogs for experiments. The miR-29a-5p analogs were purchased from Guangzhou Ruibo Company, and the cells were transfected using Invitrogen's Lipofectamine RNAiMAX transfection reagent. The transfection steps were performed according to the instructions, and the concentration of miR-29a-5p analogs was 50 nM.

[0034] A NC control group (miR-NC) was set up and transfected with the same concentration of mimic control (purchased from Guangzhou Ruibo Company).

[0035] 3. Proliferation Detection

[0036] Real-time quantitative PCR was used to detect cell proliferation marker molecules, cell growth curves were drawn, cell cycle was detected by flow cytometry, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation experiments were performed as follows:

[0037] (1) Real-time quantitative PCR detection of cell proliferation markers

[0038] 24 h after transfection of miR-29a-5p analogs, RNA was extracted from 6-well plate cells using Trizol method, RNA concentration and purity were determined by NanoDrop 2000, reverse transcribed into cDNA using reverse transcription kit (Thermo), and real-time quantitative PCR was performed using SYBR Green MasterMix (Roche).

[0039] The primers are as follows:

[0040] Cdk2-F (SEQ ID NO.2): 5'-CTTTGCCGAAATGGTGACCC-3';

[0041] Cdk2-R (SEQ ID NO.3): 5'-CCCAGAGTCCGAAAGATCCG-3';

[0042] Ccnd1-F (SEQ ID NO.4): 5'-TCAAGTGTGACCCGGACTG-3';

[0043] Ccnd1-R (SEQ ID NO.5): 5'-GGATCGATGTTCTGCTGGGC-3';

[0044] Pcna-F (SEQ ID NO.6): 5'-ATATGCCGGGACCTTAGCCA-3',;

[0045] Pcna-R (SEQ ID NO.7): 5'-TCGCAGAAAACTTCACCCCG-3';

[0046] Gapdh-F (SEQ ID NO.8): 5'-CCACGGCAAGTTCAACGGCACAG-3';

[0047] Gapdh-R (SEQ ID NO. 9): 5'-GACGCCAGTAGACTCCACGACAT-3'.

[0048] Among them, Cdk2 (Cyclin dependent kinase 2), Ccnd1 (Cyclin D1) and Pcna (Proliferating cell nuclear antigen) are cell proliferation marker molecules, and Gapdh (Glyceraldehyde-3-phosphate dehydrogenase) is an internal reference.

[0049] The results are as follows Figure 1As shown in A, miR-29a-5p analogs can significantly inhibit the expression of cell proliferation marker molecules, that is, inhibit cell proliferation.

[0050] (2) Draw cell growth curve

[0051] MTS (Promega) is a new tetrazolium compound that can be reduced by cells into colored products. It is completed in metabolically active cells and can therefore be used for cell activity detection. On days 0, 1, 2, 3, and 4 after transfection of miR-29a-5p analogs, 20 μL of MTS solution was added to each well of the 96-well plate cells. After incubation at 37°C for 3 hours, the absorbance at 490 nm was detected. The corresponding cell number was calculated using the standard curve to draw a growth curve.

[0052] The results are as follows Figure 1 As shown in B, miR-29a-5p analogs can significantly inhibit cell growth.

[0053] (3) Flow cytometry detection of cell cycle

[0054] 24 hours after transfection of miR-29a-5p analogs, the 100 mm culture dish cells were digested with 0.25% trypsin to collect neural stem cells, washed with pre-cooled PBS, fixed with pre-cooled 75% ethanol for 24 hours, and then used BD Pharmingen TM The cells were stained with PI / RNase solution for 30 min, and finally the cell cycle distribution was detected using a cell flow cytometer (BD Biosciences).

[0055] The results are as follows Figure 1 As shown in C, miR-29a-5p analogs can significantly reduce the proportion of cells in the S phase (DNA replication phase) and increase the proportion of cells in the G1 phase, that is, they can inhibit cell proliferation.

[0056] (4) EdU incorporation experiment

[0057] EdU is a thymidine analog that can be incorporated into replicating DNA molecules and can therefore be used to detect cell DNA replication activity. 24 hours after transfection of miR-29a-5p analogs, EdU was added to the cell culture medium (final concentration 50 μM) and incubated at 37°C for 2 hours. The EdU detection kit instructions were followed and the EdU positive cell ratio was obtained by counting the number of positive signal cells and the total number of cells in at least 3 fields of view.

[0058] The results are as follows Figure 1 As shown in D, miR-29a-5p analogs can significantly reduce the proportion of EdU-positive cells, indicating that they can inhibit cell DNA replication, that is, inhibit proliferation.

[0059] 4. Differentiation detection

[0060] Cell differentiation markers were detected by real-time quantitative PCR, protein immunoblotting, immunofluorescence, flow cytometry, etc., as follows:

[0061] (1) Real-time quantitative PCR detection of cell differentiation marker molecules

[0062] 24 h after transfection of miR-29a-5p analogs, RNA was extracted from 6-well plate cells using Trizol method, RNA concentration and purity were determined by NanoDrop 2000, reverse transcribed into cDNA using reverse transcription kit (Thermo), and real-time quantitative PCR was performed using SYBR Green MasterMix (Roche).

[0063] The primers are as follows:

[0064] Map2-F (SEQ ID NO.10): 5'-CTTGATTCTATTGCCCTTGGGTTTA-3';

[0065] Map2-R (SEQ ID NO. 11): 5'-CATCCATCGTTCCGCTAGTGTTG-3';

[0066] Syn1-F (SEQ ID NO.12): 5'-CCTCATTCGTGCTGCCTGTGGTA-3';

[0067] Syn1-R (SEQ ID NO.13): 5'-CTGGGCCTTTGCTTGTTTATTTT-3';

[0068] Neurod1-F (SEQ ID NO.14): 5'-CAGGGTTATGAGATCGTCACTATTC-3';

[0069] Neurod1-R (SEQ ID NO. 15): 5'-CCTTCTTGTCTGCCTCGTGTTCC-3';

[0070] Stmn2-F (SEQ ID NO.16): 5'-CACTTGGAGACTGTGAGCTGGTT-3';

[0071] Stmn2-R (SEQ ID NO. 17): 5'-ATTGCCCTATGGGAATGAAA-3';

[0072] Gfap-F (SEQ ID NO. 18): 5'-CTCAAGAGGAACACTGTGGTAAAGA-3';

[0073] Gfap-R (SEQ ID NO. 19): 5'-CTGAACGAGCCGTGGGCATAAAA-3';

[0074] Gapdh-F (SEQ ID NO.8): 5'-CCACGGCAAGTTCAACGGCACAG-3';

[0075] Gapdh-R (SEQ ID NO. 9): 5'-GACGCCAGTAGACTCCACGACAT-3'.

[0076] Among them, Map2 (Microtubule-associated protein 2), Syn1 (Synapsin I), Neurod1 (Neuronal differentiation 1) and Stmn2 (Stathmin2) are neuron marker molecules, Gfap (Glialfibrillary acidic protein) is an astrocyte marker molecule, and Gapdh is an internal reference.

[0077] The results are as follows Figure 2 As shown in A, miR-29a-5p analogs can significantly increase the expression of neuronal marker molecules and reduce the expression of astrocyte marker molecules, that is, they can promote the differentiation of neural stem cells into neurons.

[0078] (2) Detection of cell differentiation markers by protein immunoblotting

[0079] 24h after transfection of miR-29a-5p analogs, cells in 100mm culture dishes were lysed with RIPA lysis buffer (with protease inhibitor PMSF), sonicated, centrifuged and supernatant was taken to determine protein concentration. SDS-polyacrylamide gel was prepared, and equal amounts of protein samples (15μg) were loaded on each well. Electrophoresis was performed at 100V first and then at 150V. After running the gel, the membrane was transferred to PVDF membrane at 20V, 2.5mA, and transferred for 7min. The membrane was washed 3 times with TBST, blocked with 5% skim milk at room temperature on a shaker for 2h, and incubated with primary antibody at 4℃ overnight. The next day, the membrane was washed 3 times with TBST, incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 2h, washed 3 times with TBST, developed with ECL, and images were collected with Chemidoc XPS system. Grayscale values ​​were analyzed with Quantity One software. The primary antibodies are as follows: anti-Map2 antibody (1:1000, Millipore), anti-Gfap antibody (1:1000, Millipore).

[0080] The results are as follows Figure 2 As shown in B, miR-29a-5p analogs can significantly increase the expression of neuronal marker molecules and reduce the expression of astrocyte marker molecules, that is, promote the differentiation of neural stem cells into neurons.

[0081] (3) Immunofluorescence detection of cell differentiation marker molecules

[0082] 24h after transfection of miR-29a-5p analogs, the 24-well plate crawling cells were washed 3 times with DPBS, fixed with 4% paraformaldehyde for 20min, washed 3 times with 0.1% BSA-PBS, added with membrane blocking solution (PBS+goat serum+Triton X-100+BSA), incubated at room temperature for 2h, and incubated with primary antibody at 4°C overnight. The next day, washed 3 times with 0.1% BSA-PBS, incubated with fluorescent secondary antibody at room temperature in the dark for 2h, washed 3 times with 0.1% BSA-PBS, stained with Hoechst (1:1000) at room temperature in the dark for 10min, washed 3 times with 0.1% BSA-PBS, sealed, and photographed with Zeiss fluorescence microscope. The primary antibodies are as follows: anti-Tuj1 antibody (1:1000, Millipore), anti-Gfap antibody (1:1000, Millipore). Tuj1 is a neuronal marker molecule.

[0083] The results are as follows Figure 2 As shown in C, miR-29a-5p analogs can significantly increase the proportion of Tuj1-positive cells, a neuronal marker molecule, thereby promoting the differentiation of neural stem cells into neurons.

[0084] (4) Flow cytometry detection of cell differentiation markers

[0085] 24 hours after transfection of miR-29a-5p analogs, cells in 100 mm culture dishes were digested and collected, washed twice with pre-cooled PBS, fixed in 1×Fix / Perm Buffer working solution at 4°C for 50 min, washed twice with 1×Perm / Wash buffer, 100 μL 1×Perm / Wash buffer was added to the cell sample, incubated with APC-conjugated anti-Tuj1 antibody (BD Biosciences) at 4°C for 50 min, washed twice with 1×Perm / Wash buffer, resuspended in 350 μL staining buffer, and detected by flow cytometer (BD Biosciences).

[0086] The results are as follows Figure 2 As shown in D, miR-29a-5p analogs can significantly increase the proportion of Tuj1-positive cells, a neuronal marker molecule, thereby promoting the differentiation of neural stem cells into neurons.

[0087] The above embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the protection scope of the present invention.

Claims

1. Application of miR-29a-5p and its analogs in the preparation of drugs for promoting the directional differentiation of neural stem cells.

2. The use according to claim 1, characterized in that: The nucleotide sequence of miR-29a-5p is shown in SEQ ID NO.

1.

3. Use of miR-29a-5p and its analogs as described in claim 1 or 2 in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

4. The use according to claim 3, characterized in that: The neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

5. The use according to claim 3, characterized in that: The miR-29a-5p and its analogs prevent and / or treat neurodegenerative diseases by promoting the directional differentiation of neural stem cells into neurons.

6. A pharmaceutical composition for treating neurodegenerative diseases, characterized in that: It comprises miR-29a-5p and its analogs as described in claim 1 or 2, and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that The auxiliary materials include one or more of diluents, excipients, fillers, adhesives, wetting agents, absorption promoters, surfactants and adsorption carriers.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is a non-oral dosage form.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is in the form of tablets, capsules, powders, pills, granules, solutions, suspensions, injections, suppositories or sprays.

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