Use of small molecule RNA hsa-miR-451a in preparation of a drug for treating ischemic stroke

Abstract

The application of small molecule RNA hsa-miR-451a in the preparation of a drug for treating ischemic stroke belongs to the field of biotechnology research. The application protects the application of small molecule RNA hsa-miR-451a in the preparation of a drug for treating ischemic stroke. The application can effectively protect cells from ischemia-induced death, reduce infarction area, and effectively improve the treatment effect of the preparation of a drug for treating ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology research, specifically relating to the application of a small molecule RNA hsa-miR-451a in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Stroke affects millions of people worldwide each year and is one of the leading causes of death [1-2]. Ischemic stroke is the most common type of stroke, accounting for 80% of all stroke cases [3]. The pathogenesis of ischemic stroke stems from insufficient blood supply to the affected brain tissue, resulting in less oxygen and glucose being received than needed. As a result, brain damage occurs, which can lead to permanent and irreversible cell death. For many years, there have been few breakthroughs in the treatment of ischemic stroke. Therefore, extensive research has focused on identifying effective therapies to save affected cells from death.

[0003] In recent years, small RNAs (microRNAs or miRNAs) have gradually gained attention from many researchers in the study of drugs for ischemic stroke [4-7]. The main reasons why miRNAs have become a research hotspot are: 1) They can regulate hundreds or thousands of genes and can regulate cells at the system level; 2) They are only 21-23 nucleotides long and can be used as easily absorbed small molecule drugs; 3) They are easier to synthesize and have a lower cost as drugs; 4) They are small molecules that cells themselves possess, which can avoid potential immune (rejection) reactions; 5) They can be released from cells into the bloodstream, making them easier to detect and diagnose in blood.

[0004] miRNA is a non-coding RNA transcribed from genes in the cell nucleus and eventually formed as a single-stranded RNA of approximately 22 nucleotides in size in the cytoplasm. miRNA can bind to the 3' untranslated region (3'UTR) of mRNA, thereby reducing protein translation [8-10]. It has been found that miRNA regulates more than 60% of coding RNAs (which are eventually translated into proteins) in mammals. These proteins participate in various aspects of cellular activity, such as development, proliferation, fate determination, growth control, and apoptosis [11-12]. Since miRNA can only recognize the 3'UTR of target mRNA through approximately 7 nucleotides at its 5' end (called the seed region), theoretically, one miRNA can bind to hundreds of mRNAs; the 3'UTR of a single mRNA can also have multiple sequences complementary to the miRNA seed region. Therefore, as a drug target, a small amount of miRNA can control multiple signaling pathways and activities at the cellular level.

[0005] Some research groups at home and abroad have carried out basic and applied research on miRNA and ischemic stroke. For example, by targeting 5-lipoxygenase to reduce cell death, miR-193b has been found to have potential neuroprotective effects in ischemic stroke

[13] . Downregulation of miR-27b can enhance AMPKα2-mediated angiogenesis after ischemic stroke, thereby promoting long-term recovery

[14] . Intraventricular injection of miR-3473b inhibitors may reduce the expression of pro-inflammatory proteins by targeting SOCS3 during ischemic stroke. The above facts indicate that: 1) miRNA has a practical application basis and research value as a drug for the treatment of ischemic stroke; 2) miRNA has entered the R&D plans of major pharmaceutical companies, and the development of new miRNAs as therapeutic drugs for ischemic stroke has good market prospects.

[0006] References

[0007] 1. Feigin VL, Nguyen G, Cercy K, Johnson CO, Alam T, Parmar PG, et al. Global, Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016. N Engl J Med. 2018; 379: 2429-2437.

[0008] 2.Feigin VL,Norrving B,Mensah GA.Global Burden of Stroke.CircRes.2017;120:439-448.

[0009] 3.Go AS,Mozaffarian D,Roger VL,Benjamin EJ,Berry JD,Blaha MJ,etal.Heart Disease and Stroke Statistics-2014 Update A Report From the AmericanHeart Association.Circulation.2014;129:E28-E292.

[0010] 4. Stanzione R, Cotugno M, Bianchi F, Marchitti S, Forte M, Volpe M, et al. Pathogenesis of Ischemic Stroke: Role of Epigenetic Mechanisms. Genes. 2020; 11:89.

[0011] 5. Stamatovic SM, Phillips CM, Martinez-Revollar G, Keep RF, AndjelkovicAV. Involvement of Epigenetic Mechanisms and Non-coding RNAs in Blood-BrainBarrier and Neurovascular Unit Injury and Recovery After Stroke. FrontNeurosci. 2019; 13:15.

[0012] 6. Xu WL, Gao LS, Zheng JW, Li T, Shao AW, Reis C, et al. The Roles of MicroRNAs in Stroke: Possible Therapeutic Targets. Cell Transplant. 2018; 27:1778-1788.

[0013] 7. Tiedt S, Dichgans M. Role of Non-Coding RNAs in Stroke. Stroke. 2018; 49: 3098-3106.

[0014] 8.Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,andfunction.Cell.2004;116:281-297.

[0015] 9. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol CellBiol. 2014; 15:509-524.

[0016] 10. Zhou Donggen, Luo Yuping, Li Siguang. Structure, biosynthesis and function of microRNA. Biotechnology Bulletin. 2005: 20-26.

[0017] 11. Friedman RC, Farh KK-H, Burge CB, Bartel DP. Most mammalian mRNAs areconserved targets of microRNAs. Genome Research. 2009; 19:92-105.

[0018] 12. Lang MF, Shi Y. Dynamic Roles of microRNAs in Neurogenesis. Frontiers in Neuroscience. 2012; 6.

[0019] 13.Chen ZH, Yang JQ, Zhong JJ, Luo Y, Du WM, Hu CL, et al. MicroRNA-193b-3palleviates focal cerebral ischemia and reperfusion-induced injury in rats byinhibiting 5-lipoxygenase expression. Exp Neurol. 2020; 327:11.

[0020] 14. Yuan Y, Zhang Z, Wang Z, Liu J. MiRNA-27b Regulates Angiogenesis by Targeting AMPK in Mouse Ischemic Stroke Model. Neuroscience. 2019; 398: 12-22. Summary of the Invention

[0021] To address the aforementioned shortcomings, this invention provides the application of the small molecule RNA hsa-miR-451a in the preparation of drugs for treating ischemic stroke. This application effectively protects cells from ischemia-induced death and reduces infarct area.

[0022] The small RNA hsa-miR-451a used in this invention to solve the technical problem has the following nucleotide sequence:

[0023] 5'-a aaccguu accauuacugaguu-3'

[0024] Among them 5'- aaccguu -3' is the seed region sequence of the small RNA hsa-miR-451a.

[0025] This invention protects the use of a small molecule RNA hsa-miR-451a in the preparation of a drug for treating ischemic stroke.

[0026] Furthermore, the application of the small molecule RNA hsa-miR-451a in the preparation of drugs that inhibit apoptosis in the treatment of ischemic stroke.

[0027] Furthermore, the application of small RNA constructed with the seed region sequence of small RNA hsa-miR-451a as the core in the preparation of drugs for treating ischemic stroke.

[0028] Furthermore, the application of small RNA constructed with the seed region sequence of hsa-miR-451a as the core in the preparation of drugs that inhibit apoptosis in the treatment of ischemic stroke.

[0029] This invention utilizes qRT-PCR technology to detect the expression of hsa-miR-451a on the first, third, and seventh days after ischemic stroke. It found that at all three time points, higher hsa-miR-451a expression was detected on the affected side of the mouse cerebral cortex, especially on the third day.

[0030] This invention observed that after injecting hsa-miR-451a into the mouse brain, higher hsa-miR-451a expression significantly reduced the volume of infarcted brain tissue, while hsa-miR-451a inhibitors increased the volume of infarcted tissue.

[0031] In this invention, TUNEL staining was performed on brain slices from MCAO mice. A lower percentage of apoptotic cells was observed in mice receiving an hsa-miR-451a mimic (with lower Phd3 and p53) compared to the control group. However, when hsa-miR-451a levels were reduced using an hsa-miR-451a inhibitor (with higher Phd3 and p53), more apoptotic cells were observed.

[0032] This invention uses qRT-PCR technology to detect Phd3 expression on the first, third, and seventh days after ischemic stroke. It found that at all three time points, lower Phd3 expression was detected on the affected side of the mouse cerebral cortex, especially on the third day, which was negatively correlated with hsa-miR-451a expression.

[0033] This invention utilizes bioinformatics methods to identify Phd3 as the target gene of hsa-miR-451a, and constructs a reporter vector for Phd3, named psiCheck-Phd3. Dual-fluorescence detection results indicate that Phd3 is indeed the target gene of hsa-miR-451a.

[0034] After transfecting hsa-miR-451a in vitro, the target gene was detected by RT-PCR and immunofluorescence. The results showed that the expression of the target gene Phd3 was downregulated at both the mRNA and protein levels.

[0035] In this invention, after injection of hsa-miR-451a into the mouse brain (Phd3 level downregulation), p53 level was significantly reduced, while administration of hsa-miR-451a inhibitor (Phd3 level upregulation) significantly increased p53 level.

[0036] Therefore, the overexpression of hsa-miR-451a in this invention may reduce the number of apoptotic cells in the brain during ischemic stroke through its target gene Phd3 and its downstream gene p53. This may be the reason why hsa-miR-451a reduces infarct volume and protects cells from death, which is of great significance to the relationship between hsa-miR-451a and ischemic stroke.

[0037] Principle: In a mouse MCAO model of ischemic stroke, higher expression of hsa-miR-451a was observed in the cerebral cortex. Intraventricular injection of an hsa-miR-451a mimic significantly reduced infarct volume in MCAO mice. In mice injected with the hsa-miR-451a mimic, TUNEL staining revealed that hsa-miR-451a protects cells from ischemia-induced death. The mechanism of hsa-miR-451a's anti-apoptotic effect was explored, and Phd3 (also known as Egln3) was identified as a target of hsa-miR-451a both in vitro and in vivo. Finally, p53 was considered a downstream target of Phd3 mediating the anti-apoptotic effect of hsa-miR-451a. This study identifies a novel role of hsa-miR-451a in ischemia-induced apoptosis and reveals a new pathway involving hsa-miR-451a-Phd3-p53. Currently, research on hsa-miR-451a has not yet been applied to the treatment of ischemic stroke. Therefore, research on the differential expression of hsa-miR-451a to treat ischemic stroke can be designed, which is of great significance for the research of drugs for treating ischemic stroke.

[0038] Beneficial effects: The application provided by this invention can effectively protect cells from ischemia-induced death, reduce infarct area, and effectively improve the therapeutic effect of drugs for treating ischemic stroke. Attached Figure Description

[0039] Figure 1 The graph shows the expression of hsa-miR-451a after MCAO detection by qRT-PCR.

[0040] Figure 2 A figure showing how hsa-miR-451a reduces the infarct area in MACO mice.

[0041] Figure 3 A diagram showing how hsa-miR-451a reduces apoptosis in MCAO mice.

[0042] Figure 4 The graph shows the expression of Phd3 after MCAO detection by qRT-PCR.

[0043] Figure 5 Dual fluorescent reporter assay for hsa-miR-451a and its hypothesized target gene Phd3; a graph showing that hsa-miR-451a reduces Phd3 mRNA expression in MCAO mice.

[0044] Figure 6 A graph showing that hsa-miR-451a reduced the expression of Phd3 protein in MCAO mice.

[0045] Figure 7 A diagram showing the expression of p53 in MCAO mice as detected by qRT-PCR. Detailed Implementation

[0046] The present invention will be further described below with reference to the embodiments.

[0047] Example 1: Detection of hsa-miR-451a expression level in brain tissue of ischemic stroke

[0048] Total RNA was extracted from ischemic stroke (MCAO) brain tissue using the Trizol method. Cells were washed twice with PBS, and 1 mL of Trizol was added to each vial. The mixture was pipetted and incubated at room temperature for 5 min. The cell suspension was transferred to DEPC-treated 1.5 mL centrifuge tubes, and 0.2 mL of chloroform was added to each tube. The tubes were vigorously vortexed for 15 s to mix, and then incubated on ice for 3–5 min to allow for layering. The tubes were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was carefully aspirated and transferred to another DEPC-treated 1.5 mL centrifuge tube. An equal volume of pre-chilled isopropanol (4 °C) was added, and the tubes were gently mixed by inverting for 15–30 s. The tubes were incubated on ice (at room temperature) for 5–10 min. The tubes were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was discarded, and 1 mL of 75% ethanol was added to gently wash the precipitate twice. The tubes were centrifuged at 7,500 rpm for 10 min at 4 °C. The supernatant was discarded, and the tubes were inverted and air-dried. 30 μL of DEPC-treated water was added to dissolve the RNA. The concentration and purity of total RNA were determined by ultraviolet absorption assay and agarose gel electrophoresis.

[0049] The total RNA extracted above was used to synthesize cDNA using a Takara reverse transcription kit. The reaction system (20 μL) was as follows: 0.1-1 μg total RNA, 4 μL 5× reverse transcription buffer, 1 μL reverse transcriptase, and EDPC water to a final volume of 20 μL. The mixture was thoroughly combined, and reverse transcription was performed on a PCR instrument under the following conditions: 37℃ for 15 min, 85℃ for 5 s, and stored at 4℃.

[0050] Using cDNA as a template, Real-time PCR was performed on a real-time quantitative PCR instrument using specific hsa-miR-451a TaqMan primers. The reaction system (20 μL) was as follows: 1 μL of 20×TaqMan MicroRNA Assays. Universal Master Mix 10 μL, cDNA 5 ng, DEPC water to 20 μL. Reaction conditions: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. Cel-39 small RNA was used as an internal control. -ΔΔCt Data is processed using relative quantification methods.

[0051] Real-time PCR results showed that, compared with normal brain tissue, higher hsa-miR-451a expression was detected in the affected side of the mouse cerebral cortex on days 1, 3, and 7 after MCAO, with the highest expression on day 3. Figure 1 ).

[0052] Example 2: hsa-miR-451a reduced the infarct area in MACO mice

[0053] Prepare an in vivo transfection solution by mixing 3.5 μL of hsa-miR-451a mimic or an hsa-miR-451a inhibitor with 3.5 μL of siRNA-Mate transfection reagent. Inject the transfection solution into the ventricle of the affected side (the side with MCAO) of C57BL / 6 mice at the following locations: anterior fontanelle: -2.5 mm, ventral: 1 mm, lateral: 1.5 mm. One day after intraventricular injection, MCAO was performed. 48 hours later, frozen sections of the mouse brain tissue were prepared. The brain sections were immersed in Nissl staining solution for 15 minutes, washed with distilled water, and incubated with 95% ethanol for 5 seconds. Nissl-stained images of the brain were loaded into Adobe Photoshop for infarct volume measurement. Figure 2 ).

[0054] Example 3: hsa-miR-451a reduced apoptosis in MCAO mice

[0055] hsa-miR-451a mimic and hsa-miR-451a inhibitor were transfected in vitro into the affected side of C57BL / 6 mice, as described in Example 2. Mouse brain tissue was frozen sections fixed with 4% paraformaldehyde for 20 min, and the cells were immersed in permeation buffer on ice for 2 min. Excess fluid around the tissue on the slide was carefully blotted off with filter paper, and 20 μL Tunnel reaction solution was immediately added to the sections. The slides were then incubated at 37°C for 1 h. Cell staining was observed under a fluorescence microscope. More apoptotic cells were observed in the mouse brain tissue transfected with hsa-miR-451a inhibitor, while fewer apoptotic cells were observed in the mouse brain tissue transfected with hsa-miR-451a mimic. Figure 3 ).

[0056] Example 4: Detection of Phd3 expression level in brain tissue of ischemic stroke

[0057] Total RNA was extracted from brain tissue using the Trizol method. The extraction method was the same as in Example 1. After detecting the concentration and purity of total RNA by UV absorbance assay and agarose gel electrophoresis, the extracted total RNA was used to synthesize cDNA using a kit.

[0058] The total RNA extracted above was used to synthesize cDNA using a Takara reverse transcription kit. The reaction system (20 μL) was as follows: 0.1 μg–1 μg total RNA, 4 μL 5× reverse transcription buffer, 1 μL reverse transcriptase, and EDPC water to a final volume of 20 μL. The mixture was thoroughly combined, and reverse transcription was performed on a PCR instrument under the following conditions: 37℃ for 15 min, 85℃ for 5 s, and stored at 4℃.

[0059] Then, using cDNA as a template, RT-PCR was performed on the target gene. The reaction system (20 μL) was as follows: 10 μL 2× reaction buffer, 0.4 μL 10 μM upstream primer, 0.4 μL 10 μM downstream primer, 1 μL cDNA, and 8.2 μL DEPC water. Reaction conditions: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, 40 cycles, stored at 4℃. β-actin was used as an internal control for the target gene.

[0060] RT-PCR results showed that, compared with the control, Phd3 expression was decreased on days 1, 3, and 7 after MCAO, with the lowest expression on day 3. Figure 4 ).

[0061] Example 5: Prediction of hsa-miR-451a target genes

[0062] Using bioinformatics methods, we predicted target genes associated with hsa-miR-451a using online databases (TargetScan: http: / / www.targetscan.org / ; miRWalk: mirwalk.umm.uni-heidelberg.de; and MiRtaget2: mirdb.org). Phd3 was tentatively identified as the target gene of hsa-miR-451a.

[0063] Example 6: Construction of hsa-miR-451a target gene reporter vector

[0064] Based on the Phd3 gene sequence in GenBank, primers (Phd3-F and Phd3-R) were designed using a sequence matching hsa-miR-451a. Phd3-F had a 5′ restriction site for the restriction enzyme XhoI and a protective base attached to its 5′ end, while Phd3-R had a 5′ restriction site for another restriction enzyme, NotI, and a protective base attached to its 5′ end. PCR amplification was then performed using the following PCR reaction system (20 μL): 4 μL 5× reaction buffer, 1 μL 10 μM upstream primer, 1 μL 10 μM downstream primer, 0.9 μL DNA (less than 1 μg), 1.6 μL 10 mM dNTPs, 0.2 μL Phusion DNA Polymerase, and 11.3 μL ddH2O. Reaction conditions: 98℃ for 30 s; 98℃ for 7 s, 67.3℃ for 20 s, 72℃ for 45 s, 30 cycles; extension at 72℃ for 7 min; storage at 4℃. After amplification, the PCR product was double-digested with the following enzyme mixture (30 μL): 3 μL 10×T Buffer, 1 μL XhoI, 1 μL Not I, and 25 μL PCR product. The digestion reaction was carried out in a 37℃ water bath. After 3 h, the digested product was recovered using a gel extraction kit and finally dissolved in 10 μL Buffer EB.

[0065] The psiCheck control vector was then subjected to double digestion with the following 10 μL digestion system: 1 μL 10×T Buffer, 1 μL XhoI, 1 μL Not I, 4 μL psiCheck control, and 3 μL ddH2O. The digestion reaction was carried out in a 37°C water bath. After 3 hours, the digestion products were recovered using a gel extraction kit and finally dissolved in 10 μL Buffer EB.

[0066] The enzyme digestion products were ligated using a ligation system (12 μL): 1.2 μL 10× Buffe ligation enzyme, 4.6 μL vector fragment, 2.3 μL target gene fragment, 1 μL T4 DNA ligase, and 2.9 μL ddH2O. The ligation reaction was carried out at room temperature for 10 min. After 16–20 h, the ligase was inactivated by incubating in a 70°C water bath for 10 min, thus terminating the ligation reaction.

[0067] The ligation product was transformed into Escherichia coli DH5α, plated on agar plates, and incubated at 37°C for 12–16 h. Positive clones were then selected and subjected to PCR amplification, restriction enzyme digestion, and sequencing verification to obtain the correct hsa-miR-451a expression vector, named psiCheck-Phd3.

[0068] Example 7: In vitro transfection of hsa-miR-451a target gene reporter vector

[0069] KEK293A cells were transfected using Invitrogen's Lipofectamine 2000 transfection reagent and Invitrogen-purchased psiCheck-Phd3 for transfection. The transfection procedure is briefly described below: One day before transfection, 4 × 10⁶ cells per well were transfected. 5 Cells were seeded at a concentration of 1,000 cells per well in 6-well plates. After 20 hours of routine culture, the cells reached the logarithmic growth phase. Appropriate volumes of psiCheck-Phd3 / psiCheck-mut-Phd3 and hsa-miR-451a / hsa-miR-451a-control were diluted in 100 μL of OPTI-MEM medium and gently mixed to a final concentration of 20 μg / mL. Lipofectamine 2000: Prepare the transfection mixture in a 96-well plate at a ratio of 4:2:psiCheck-Phd3+hsa-miR-451a / psiCheck-mut-Phd3+hsa-miR-451a-control and shake vigorously for 5-10 seconds. After standing for 15 minutes, slowly add the transfection mixture to the wells containing KEK293A cells and OPTI-MEM medium and shake gently for 30 seconds. Incubate at 37°C with 5% CO2 for 6 hours, then replace with KEK293A cell medium and continue culturing for 48-72 hours before detecting the transfection level and conducting subsequent experiments.

[0070] Example 8: In vivo and in vitro detection of hsa-miR-451a's direct targeting inhibition of Phd3

[0071] (1) Detection of dual fluorescence reporting

[0072] KEK293A cells were transfected 24 h after seeding. The transfection method was the same as in Example 7, followed by dual-luciferase reporter assay. The dual-luciferase reporter assay was performed using the Promega Dual-luciferase reporter gene assay system, following the kit instructions. A brief procedure is as follows: 48 h after transfection, KEK293A cells were washed once with PBS, then lysed with 100 μL of passive lysis buffer in each well. 15 μL of the lysate was added to 50 μL of Luciferase reaction substrate, and Firefly fluorescence was measured using a fluorescence meter. Then, 50 μL of StopGlo reagent was added, and Renilla fluorescence was immediately measured. The fluorescence values ​​of Firefly and Renilla luciferase were analyzed and normalized to the Firefly fluorescence value using the Renilla luciferase fluorescence value. Each experiment was repeated at least three times, and the average value was taken.

[0073] Dual-fluorescence reporter assay results showed that when the hsa-miR-451a target gene reporter vector Phd3-WT-3'UTR was co-transfected with hsa-miR-451a into KEK293A cells, the fluorescence value of luciferase was significantly reduced, indicating that Phd3 is indeed a target gene of hsa-miR-451a. Figure 5 ).

[0074] (2) RT-PCR detection of differential expression of target genes at the mRNA level

[0075] hsa-miR-451a mimic and hsa-miR-451a inhibitor were transfected in vitro into the affected side of C57BL / 6 mice, as described in Example 2. Total RNA was extracted from the cells using the Trizol method. The extraction method was the same as in Example 1. After detecting the concentration and purity of total RNA by UV absorbance assay and agarose gel electrophoresis, the extracted total RNA was used to synthesize cDNA using a kit.

[0076] The total RNA extracted above was used to synthesize cDNA using a Takara reverse transcription kit. The reaction system (20 μL) was as follows: 0.1 μg–1 μg total RNA, 4 μL 5× reverse transcription buffer, 1 μL reverse transcriptase, and EDPC water to a final volume of 20 μL. The mixture was thoroughly combined, and reverse transcription was performed on a PCR instrument under the following conditions: 37℃ for 15 min, 85℃ for 5 s, and stored at 4℃.

[0077] Then, using cDNA as a template, RT-PCR was performed on the target gene. The reaction system (20 μL) was as follows: 10 μL 2× reaction buffer, 0.4 μL 10 μM upstream primer, 0.4 μL 10 μM downstream primer, 1 μL cDNA, and 8.2 μL DEPC water. Reaction conditions: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, 40 cycles, stored at 4℃. β-actin was used as an internal control for the target gene.

[0078] RT-PCR results showed that, compared with the control, the expression of the target gene Phd3 in hsa-miR-451a transfected cells was reduced at the mRNA level. Figure 5 ).

[0079] Example 9: Immunofluorescence detection of differential expression of target genes at the protein level

[0080] The differential expression of target genes at the protein level was detected using immunofluorescence. The detection steps are briefly described below:

[0081] Prepare an in vivo transfection solution by mixing 3.5 μL of hsa-miR-451a mimic or an hsa-miR-451a inhibitor with 3.5 μL of siRNA-Mate transfection reagent. Inject the transfection solution into the ventricle of the affected side (the side with MCAO) of C57BL / 6 mice, and prepare frozen sections using the same method as in Example 2. Incubate the frozen sections of mouse brains with 0.3% Triton X-100 and 5% goat serum in PBS for 30 min, then incubate overnight with primary antibody, and finally incubate with secondary antibody at room temperature for 4 h. The primary antibody was rabbit anti-Phd3 (1:200), and the secondary antibody was Cy3-conjugated goat anti-rabbit IgG (1:400). Cell nuclei were stained with DAPI.

[0082] Immunofluorescence results showed that, compared with the control, the protein expression of the target gene Phd3 in patients transfected with hsa-miR-451a was significantly reduced. Figure 6 ).

[0083] Example 10: Detection of p53 expression levels in brain tissue

[0084] hsa-miR-451a mimic and hsa-miR-451a inhibitor were transfected in vitro into the affected side of C57BL / 6 mice, as described in Example 2. Total RNA was extracted from the cells using the Trizol method. The extraction method was the same as in Example 1. After detecting the concentration and purity of total RNA by UV absorbance assay and agarose gel electrophoresis, the extracted total RNA was used to synthesize cDNA using a kit.

[0085] The reaction mixture (20 μL) consisted of: 100 pg-100 g total RNA, 4 μL 5× reverse transcription buffer, 2 μL 10× SuperScript reverse transcriptase, and EDPC water to a final volume of 20 μL. The mixture was thoroughly mixed, and reverse transcription was performed on a PCR instrument under the following conditions: 42℃ for 30 min, 95℃ for 5 min, and stored at 4℃.

[0086] Then, using cDNA as a template, RT-PCR was performed on the target gene. The reaction system (20 μL) was as follows: 10 μL 2× reaction buffer, 0.4 μL 10 μM upstream primer, 0.4 μL 10 μM downstream primer, 1 μL cDNA, and 8.2 μL DEPC water. Reaction conditions: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, 40 cycles, stored at 4℃. β-actin was used as an internal control for the target gene.

[0087] RT-PCR results showed that, compared with the control, p53 levels were significantly reduced after injection of hsa-miR-451a into the mouse brain (Phd3 level downregulation), while administration of an hsa-miR-451a inhibitor (Phd3 level upregulation) significantly increased p53 levels. Figure 7 ).

[0088] The above embodiments are merely illustrative and explanatory of the present invention and are not intended to limit the invention to the scope of the described embodiments. Furthermore, those skilled in the art will understand that the present invention is not limited to the above embodiments, and many more variations and modifications can be made based on the teachings of the present invention, all of which fall within the scope of protection claimed by the present invention.

Claims

1. The application of a small molecule RNA hsa-miR-451a in the preparation of a drug for treating ischemic stroke, characterized in that, The hsa-miR-451a nucleotide sequence is as follows: 5'-aaaccguuaccauuacugaguu-3'; The small molecule RNA hsa-miR-451a is used to prepare a drug that inhibits apoptosis in the treatment of ischemic stroke.

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