miR-21-5p Alleviates OGD/R-induced Inflammation and Apoptosis of HT22 Cells and Promotes Proliferation by Inhibiting STAT3
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摘要:
目的 探究miR-21-5p对 OGD诱导的HT22细胞的影响机制。 方法 OGD/R诱导HT22细胞构建细胞模型。CCK-8检测细胞活力，流式细胞术检测细胞凋亡。蛋白印迹检测STAT3的表达。RT-qPCR检测炎症因子的mRNA的相对表达量。Starbase数据库预测miR-21-5p和STAT3的结合位点双荧光素酶实验验证miR-21-5p与STAT3的靶向关系。 结果 miR-21-5p在OGD/R诱导的神经元细胞中降低（P < 0.01）， p-STAT3/ STAT3在OGD/R诱导的神经元细胞中增加（P < 0.01）。miR-21-5p能靶向调控STAT3的表达。增加miR-21-5p的表达能抑制细胞凋亡和炎症，促进增殖。而Stattic能逆转miR-21-5p的作用。 结论 miR-21-5p通过抑制STAT3缓解OGD诱导的HT22细胞炎症和凋亡，促进增殖。 Abstract:Objective To investigate the effect of miR-21-5p on the injury of OGD/R-induced HT22 cells. Methods OGD / R induced HT22 cells to construct a cell model. The Cell Counting Kit-8（CCK-8） and flow cytometry were used to assess cell proliferation vitality and cell apoptosis. Western blot analysis was used to check STAT3 protein expression, and RT-qPCR was used to detect inflammatory factors expression.The Starbase database predicted the binding sites of miR-21-5p and STAT3. The dual luciferase assay verified the targeting relationship between miR-21-5p and STAT3. Results miR-21-5p was significantly decreased in OGD / R-induced neuronal cells, and p-STAT3 / STAT3 was significantly increased in OGD / R-induced neuronal cells. MiR-21-5p could target the expression of STAT3. Increased expression of miR-21-5p could significantly inhibit apoptosis and inflammation, and promote proliferation. Stattic could reverse the effect of miR-21-5p. Conclusion miR-21-5p alleviates OGD-induced inflammation and apoptosis in HT22 cells by inhibiting STAT3 and promotes proliferation. -
Key words:
- miR-21-5p /
- STAT3 /
- Cerebral ischemia /
- Inflammation /
- Apoptosis
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图 1 OGD/R诱导对HT22细胞中miR-21-5p和STAT3表达水平的影响
A：CCK-8检测细胞活力；B：流式细胞术检测细胞凋亡率；C：RT-qPCR检测miR-21-5p的相对表达量；D：蛋白印迹检测p-STAT3和STAT3的相对表达量。**P < 0.01。
Figure 1. Effects of OGD/R induction on the expression levels of miR-21-5p and STAT3 in HT22 cells
图 2 miR-21-5p影响脑缺血中神经元细胞炎症和凋亡
A：RT-qPCR检测miR-21-5p的转染效率；B：CCK-8检测细胞活力；C-1：细胞凋亡率统计结果；C-2：流式细胞仪检测结果；D：RT-qPCR检测炎症因子的mRNA相对表达水平。与NC组比较，**P < 0.01；与OGD/R组比较，##P < 0.01。
Figure 2. miR-21-5p affected neuronal cell inflammation and apoptosis in cerebral ischemia
图 3 miR-21-5p能够靶向调控STAT3
A：Starbase数据库预测miR-21-5p和STAT3的结合位点; B：双荧光素酶实验验证miR-21-5p和STAT3的靶向关系；C：蛋白印迹检测STAT3的表达。**P < 0.01。
Figure 3. miR-21-5p could target and regulate STAT3
图 4 miR-21-5p通过STAT3影响脑缺血中神经元细胞炎症和凋亡
A：CCK-8检测细胞活力；B:RT-qPCR检测miR-21-5p的相对表达量。C:流式细胞术检测细胞凋亡率；D：RT-qPCR检测炎症因子的mRNA相对表达水平。与NC组相比，** P < 0.01；与OGD/R组相比，##P < 0.01。与miR-21-5p mimic相比，&P < 0.05，&&P < 0.01。
Figure 4. miR-21-5p affected neuronal cell inflammation and apoptosis in cerebral ischemia via STAT3
表 1 引物序列
Table 1. Gene name and primer sequence
基因 序列（5′ - 3'） TNF-α-F
TNF-α-R5′-ACCCTCACACTCAGATCATCTTC-3'
5′-TGGTGGTTTGCTACGACGT-3'IL-1β-F 5′-AACCTGCTGGTGTGTGACGTTC-3' IL-1β-R 5′-CAGCACGAGGCTTTTTTGTTGT-3' IL-6-F 5′-AGAAGGAGTGGCTAAGGACCAA-3' IL-6-R 5′-ACGCACTAGGTTTGCCGAGTA-3' GAPDH-F 5′-CGGAGTCAACGGATTTTTGTAT-3' GAPDH-R 5′-AGCCTTCTCCATGGTGGTGAAGAC-3 miR-21-5p-F 5′-TCGCTCGAGATTTTTTTTTATCAAGAGGG-3' miR-21-5p-R 5′-TCGGCGGCCGCGACAAGAATGAGACTTTAATC-3' U6-F 5′-GCTTCGGCAGCACATATAAAAT-3' U6-R 5′-CGCTTCACGAATTTGCGTGTCAT-3' 
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[1] Campbell B,De Silva D,Macleod M,et al. Ischaemic stroke[J]. Nat Rev Dis primers,2019,5(1):70-91. doi: 10.1038/s41572-019-0118-8 [2] Roger V L,Go A S,Lloyd-Jones D M,et al. Heart disease and stroke statistics-2012 update: A report from the American heart association[J]. Circulation,2012,125(1): e2-e220. [3] Wu X X,Lin L,Qin J J,et al. CARD3 promotes cerebral ischemia-reperfusion injury via activation of TAK1[J]. J. Am. Heart Assoc,2020,9(9):e014920. doi: 10.1161/JAHA.119.014920 [4] Bartel D P. Metazoan microRNAs[J]. Cell,2018,173(1):20-51. doi: 10.1016/j.cell.2018.03.006 [5] Du K,Zhao C,Wang L,et al. MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1[J]. Aging,2019,11(9):2762-2786. doi: 10.18632/aging.101948 [6] Li W Y,Zhu Q B,Xu X Y,Hu X. MiR-27a-3p suppresses cerebral ischemia-reperfusion injury by targeting FOXO1[J]. Aging,2021,13(8):11727-11737. [7] Xie K,Mo Y,Yue E L,et al. Exosomes derived from M2-type microglia ameliorate oxygen-glucose deprivation/reoxygenation-induced HT22 cell injury by regulating miR-124-3p/NCOA4-mediated ferroptosis[J]. Heliyon,2023,9(7):e17592. doi: 10.1016/j.heliyon.2023.e17592 [8] Pan J J,Qi L,Wang L P,et al. M2 microglial extracellular vesicles attenuated blood-brain barrier disruption via miR-23a-5p in cerebral ischemic mice[J]. Aging Dis,2024,15(3):1344-1356. [9] Cao X Y,Zhang X J,Chen J M,et al. miR-100-5p activation of the autophagy response through inhibiting the mTOR pathway and suppression of cerebral infarction progression in mice[J]. Aging (Albany NY),2023,15(16):8315-8324. doi: 10.18632/aging.204971 [10] Zhan L,Mu Z,Jiang H,et al. MiR-21-5p protects against ischemic stroke by targeting IL-6R[J]. Ann Transl Med,2023,11(2):101-117. [11] Zhang Y,Ye P,Zhu H,et al. Neutral polysaccharide from Gastrodia elata alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis-mediated neuroinflammation via the NRF2/HO-1 signaling pathway[J]. CNS Neurosci Ther,2024,30(3):e14456. doi: 10.1111/cns.14456 [12] Winkle M,El-Daly S M,Fabbri M,et al. Noncoding RNA therapeutics - challenges and potential solutions[J]. Nat Rev Drug Discov,2021,20(8):629-651. doi: 10.1038/s41573-021-00219-z [13] Hu Z Q,Li L B,Li M,et al. miR-21-5p inhibits ferroptosis in hepatocellular carcinoma cells by regulating the AKT/mTOR signaling pathway through MELK[J]. J Immunol Res,2023,2023(1):8929525. [14] Gao P,Wang H P,Li H X,et al. miR-21-5p inhibits the proliferation,migration,and invasion of glioma by targeting S100A10[J]. J Cancer,2023,14(10):1781-1793. doi: 10.7150/jca.84030 [15] He Q L,Ye A H,Ye W B,et al. Cancer-secreted exosomal miR-21-5p induces angiogenesis and vascular permeability by targeting KRIT1[J]. Cell Death Dis,2021,12(6):576-589. [16] La Manna S,De Benedictis I,Marasco D. Proteomimetics of natural regulators of JAK-STAT pathway: Novel therapeutic perspectives[J]. Front. Mol. Biosci,2021,8:792546. [17] Luo L X,Deng L Y,Chen Y T,et al. Identification of lipocalin 2 as a ferroptosis-related key gene associated with hypoxic-ischemic brain damage via STAT3/NF-κB signaling pathway[J]. Antioxidants (Basel),2023,12(1):186-203. doi: 10.3390/antiox12010186 [18] Egwuagu C E. STAT3 in CD4+ T helper cell differentiation and inflammatory diseases[J]. Cytokine,2009,47(3):149-156. doi: 10.1016/j.cyto.2009.07.003 [19] Ni H J,Liao Y F,Zhang Y F,et al. Levistilide A ameliorates neuroinflammation via inhibiting JAK2/STAT3 signaling for neuroprotection and cognitive improvement in scopolamine-induced Alzheimer's disease mouse model[J]. Int. Immunopharmacol,2023,124:110783 . [20] Ding W,Cai C,Zhu X M,et al. Parthenolide ameliorates neurological deficits and neuroinflammation in mice with traumatic brain injury by suppressing STAT3/NF-κB and inflammasome activation[J]. Int. Immunopharmacol,2022,108:108913. doi: 10.1016/j.intimp.2022.108913 -
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