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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