miR-21-5p Targetes STAT3 Reduce the OGD/R-induced Neuronal Injury
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摘要:
目的 探讨miR-21-5p对OGD/R诱导的HT22细胞损伤的保护作用和潜在机制。 方法 OGD/R诱导HT22细胞构建脑缺血再灌注损伤（cerebral ischemia reperfusion injury，CIRI）细胞模型。RT-qPCR检测miR-21-5p的表达。CCK-8法、TUNEL染色和流式细胞术分别检测细胞活力和细胞凋亡情况。ELISA检测细胞上清液中炎症因子IL-6、IL-10、TNF-α的含量。Western blot检测p-STAT3/STAT3、Cleaved-Caspase-3、Bax和Bcl-2蛋白的表达情况。TargetScan数据库预测miR-21-5p和STAT3的结合位点，双荧光素酶报告基因实验验证miR-21-5p与STAT3的靶向关系。 结果 miR-21-5p的表达水平在OGD/R诱导的HT22细胞中表达下调（P < 0.001）。OGD/R诱导后HT22细胞增殖活力下降（P < 0.0001 ）；细胞凋亡率升高（P < 0.001）；促炎因子IL-6（P < 0.001）和TNF-α（P < 0.001）的含量升高，抑炎因子IL-10（P < 0.001）含量减少；miR-21-5p mimic转染后提高了细胞活力、减少了凋亡率、抑制了神经炎症。miR-21-5p能够靶向结合STAT3。miR-21-5p inhibitor转染后降低了细胞活力、促进了细胞凋亡和神经炎症的发生，STAT3抑制剂Stattic能够逆转miR-21-5p inhibitor的作用。结论 miR-21-5p能够靶向结合STAT3，减少OGD/R诱导的HT22细胞的神经炎症和细胞凋亡。 Abstract:Objective To investigate the potential mechanism of miR-21-5p in alleviating cerebral ischemia-reperfusion injury by targeting STAT3. Methods The HT22 cells were induced by OGD/R to construct a cell model of cerebral ischemia reperfusion injury. The expression of miR-21-5p was detected by RT-qPCR. The CCK-8 assay, TUNEL staining and flow cytometry were respectively used to detect the cell viability and apoptosis. ELISA assay was used to determine the contents of inflammatory factors IL-6, IL-10 and TNF-α in the cell supernatant. Western blot was used to detect the expression levels of p-STAT3/STAT3, Cleaved-Caspase-3, Bax and Bcl-2 proteins. The TargetScan database was used to predict the binding sites of miR-21-5p and STAT3. The dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-21-5p and STAT3. Results The relative expression level of miR-21-5p was down-regulated in HT22 cells which induced by OGD/R （P < 0.001）. The cell viability （P < 0.0001 ） was decreased and the apoptosis rate （P < 0.001） was increased in OGD/R induced-HT22 cells. The contents of pro-inflammatory factors IL-6 （P < 0.001） and TNF-α （P < 0.001） was increased, while the content of anti-inflammatory factor IL-10 （P < 0.001） decreased. After transfection with miR-21-5p mimic, cell viability was enhanced, apoptosis rate was reduced and neuroinflammation was inhibited. MiR-21-5p could target and bind to STAT3. After miR-21-5p inhibitor transfection, cell viability decreased, apoptosis was promoted, and neuroinflammation occurred; STAT3 inhibitor Stattic could reverse the effect of miR-21-5p inhibitor.Conclusion MiR-21-5p could specifically bind to STAT3 and reduce the neuroinflammation and apoptosis of OGD/R induced-HT22 cells. -
图 1 OGD/R对HT22细胞的影响
A：CCK-8试剂盒检测细胞活力；B～C：TUNEL染色检测HT22细胞凋亡情况（比例尺50 μm）；D：RT-qPCR检测HT22细胞中miR-21-5p的表达情况；与正常对照组相比，***P < 0.001，****P < 0.0001。
Figure 1. Effect of OGD/R on HT22 cells
图 2 miR-21-5p mimic对OGD/R诱导的HT22细胞的影响
A：RT-qPCR检测miR-21-5p的表达情况；B：CCK-8试剂盒检测细胞活力；C～E：ELISA试剂盒检测炎症因子IL-6、TNF-α、IL-10的含量；F-G：流式细胞术检测细胞凋亡情况；H-K：Western blot检测凋亡相关蛋白Cleaved-Caspase-3、Bax和Bcl-2的表达情况；*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 2. Effect of miR-21-5p mimic on OGD/R-induced HT22 cells
图 3 miR-21-5p 靶向调控STAT3
A：STAT3与miR-21-5p的结合位点示意；B：双荧光素酶报告基因实验检测荧光素酶相对活性；C：RT-qPCR检测miR-21-5p的表达情况；D～E：Western blot检测p-STAT3和STAT3的表达情况；nsP > 0.05；*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 3. miR-21-5p targets STAT3
图 4 抑制STAT3逆转了miR-21-5p inhibitor的作用
A：RT-qPCR检测miR-21-5p的表达情况；B：RT-qPCR检测STAT3的表达情况；C～D：Western blot检测STAT3的表达情况；E-G. ELISA试剂盒检测炎症因子IL-6、TNF-α、IL-10的表达；J～M. Western blot检测凋亡相关蛋白的表达情况；*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 4. miR-21-5p targets STAT3
表 1 基因名称及引物序列
Table 1. Gene name and primer sequence
基因 引物序列 （F：正向引物；R：反向引物） （5'-3'） miR-21-5p F：TCGCTCGAGATTTTTTTTTATCAAGAGGG
R：TCGGCGGCCGCGACAAGAATGAGACTTTAATCU6 F：GCTTCGGCAGCACATATAAAAT
R：CGCTTCACGAATTTGCGTGTCAT
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