miR-135b靶向SIRT1表达对H_(2)O_(2)诱导神经细胞损伤增殖、凋亡的影响及作用机制

Effect and Mechanism of MiR-135b-targeted SIRT1 Expression on Proliferation and Apoptosis of H_(2)O_(2)-induced Neuronal Cell Injury

目的探讨miR-135b靶向沉默信息调节因子1(SIRT1)表达对过氧化氢(H_(2)O_(2))诱导神经细胞损伤增殖和凋亡的影响及作用机制。方法将HT22细胞分为对照组(正常培养的HT22细胞)、H_(2)O_(2)组、H_(2)O_(2)抑制剂阴性对照组、H_(2)O_(2)+miR-135b抑制剂组(转染miR-135b抑制剂)、H_(2)O_(2)+SIRT1过表达组(转染SIRT1过表达载体)、H_(2)O_(2)+空载体对照组(转染空载体)、H_(2)O_(2)+miR-135b抑制剂阴性对照组(转染miR-135b抑制剂阴性对照组)、H_(2)O_(2)+miR-135b抑制剂+SIRT1抑制剂组(转染miR-135b抑制剂和SIRT1抑制剂),各组均在转染后经200μmol/L的H_(2)O_(2)作用24 h。采用Western Blot检测miR-135b、SIRT1信使RNA(mRNA)、SIRT1蛋白表达水平,MTT、流式细胞仪检测细胞增殖、凋亡情况,双荧光素酶报告基因实验验证miR-135b与SIRT1之间的调控关系。结果与H_(2)O_(2)抑制剂阴性对照组相比,H_(2)O_(2)+miR-135b抑制组miR-135b水平显著降低(0.41±0.05比2.33±0.16)(P<0.05),存活率显著升高[(74.1±5.6)%比(48.3±4.1)%](P<0.05),凋亡率显著降低[(15.4±1.3)%比(33.4±2.6)%](P<0.05)。双荧光素酶报告基因结果显示,SIRT1野生型质粒(SIRT1-WT)+mimic-阴性对照组、SIRT1-WT+mimic组、SIRT1突变型质粒(SIRT1-MUT)+mimic-阴性对照组、SIRT1-MUT+mimic组双荧光素酶活性分别为1.15±0.09、0.56±0.03、1.08±0.08、1.12±0.10,各组荧光素酶活性比较差异有统计学意义(P<0.01),其中SIRT1-WT+mimic组荧光素酶活性显著低于SIRT1-WT+mimic-阴性对照组(P<0.05)。miR-135b inhibitor组SIRT1 mRNA、SIRT1蛋白水平高于inhibitor-control组(P<0.05),miR-135b mimic组SIRT1 mRNA、SIRT1蛋白水平低于mimic-control组(P<0.05)。与H_(2)O_(2)+空载体对照组相比,H_(2)O_(2)+SIRT1过表达组SIRT1 mRNA和蛋白水平显著升高(1.15±0.06比0.54±0.03、1.13±0.11比0.57±0.04)(P<0.05),存活率显著升高[(75.3±5.5)%比(45.4±3.4)%](P<0.05),凋亡率显著降低[(15.5±1.2)%比(39.4±2.8)%](P<0.05)。与H_(2)O_(2)+miR-135b抑制剂阴性对照组相比,H_(2)O_(2)+miR-135b抑制剂+SIRT1抑制剂组SIRT1蛋白水平显著降低(0.61±0.04比1.12±0.07)(P<0.05),存活率显著降低[(48.3±4.4)%比(69.2±5.4)%](P<0.05),凋亡率显著升高[(36.2±3.6)%比(13.4±1.0)%](P<0.05)。结论抑制miR-135b表达可促进H_(2)O_(2)诱导的HT22细胞增殖,抑制其凋亡,其机制可能是通过负调控SIRT1表达发挥作用。

Objective To explore the effect and mechanism of miR-135b-targeted silent information regulator 1(SIRT1)expression on proliferation and apoptosis of H_(2)O_(2)-induced neuronal cell injury.Methods HT22 cells were divided into a control group(normally cultured HT22 cells),an H_(2)O_(2)group,an H_(2)O_(2)inhibitor-negative control group,an H_(2)O_(2)+miR-135b inhibitor group(transfected with miR-135b inhibitor),an H_(2)O_(2)+SIRT1 overexpression group(SIRT1 overexpression vector),an H_(2)O_(2)+empty vector control group(transfected with empty vector),an H_(2)O_(2)+miR-135b inhibitor negative control group(miR-135b inhibitor negative control),an H_(2)O_(2)+miR-135b inhibitor+SIRT1 inhibitor group(transfection of miR-135b inhibitor and SIRT1 inhibitor).All groups were subjected to H_(2)O_(2)at 200μmol/L for 24 h after transfection.Western Blot was adopted to detect miR-135b,SIRT1 messenger RNA(mRNA),SIRT1 protein expression level,and MTT,flow cytometry were adopted to detect cell proliferation,apoptosis,and dual luciferase reporter gene assay was adopted to verify the regulatory relationship between miR-135b and SIRT1.Results Compared with the H_(2)O_(2)inhibitor-negative control group,miR-135b levels of the H_(2)O_(2)+miR-135b inhibitor group was significantly lower(0.41±0.05 vs 2.33±0.16)(P<0.05),and the survival rate significantly increased[(74.1±5.6)%vs(48.3±4.1)%](P<0.05),and apoptosis rate significantly decreased[(15.4±1.3)%vs(33.4±2.6)%](P<0.05).According to the dual-luciferase reporter results,the dual luciferase activities of the SIRT1 wild-type plasmid(SIRT1-WT)+mimic-negative control group,SIRT1-WT+mimic group,SIRT1 mutant plasmid(SIRT 1-MUT)+mimic-negative control group were 1.15±0.09,0.56±0.03,1.08±0.08 and 1.12±0.10 respectively,and there were significant differences in luciferase activity between different groups(P<0.01),among which luciferase activity in the SIRT1-WT+mimic group was significantly lower than that in the SIRT1-WT+mimic-negative control group(P<0.05).The level of SIRT1 mRNA and SIRT1 proteins in the miR-135b inhibitor group were higher than those in the inhibitor-control group(P<0.05),and the level of SIRT1 mRNA and SIRT1 proteins in the miR-135b mimic group were lower than those in the mimic-control group(P<0.05).Compared with the H_(2)O_(2)+empty vector control group,the SIRT1 mRNA and protein levels were significantly increased in the H_(2)O_(2)+SIRT1 overexpression group(1.15±0.06 vs 0.54±0.03,1.13±0.11 vs 0.57±0.04)(P<0.05),the survival rate was significantly higher[(75.3±5.5)%vs(45.4±3.4)%](P<0.05),the apoptotic rate was significantly reduced[(15.5±1.2)%vs(39.4±2.8)%](P<0.05).Compared to the H_(2)O_(2)+miR-135b inhibitor-negative control group,SIRT1 protein decreased in the H_(2)O_(2)+miR-135b inhibitor+SIRT1 inhibitor group(0.61±0.04 vs 1.12±0.07)(P<0.05),the survival rate was significantly reduced[(48.3±4.4)%vs(69.2±5.4)%](P<0.05),the apoptotic rate was significantly increased[(36.2±3.6)%vs(13.4±1.0)%](P<0.05).Conclusion Inhibition of miR-135b expression promotes H_(2)O_(2)-induced HT22 cell proliferation and inhibits its apoptosis,the mechanism of which may be through the negative regulation of SIRT1 expression.