miR-27a-3p在氧糖缺失-复氧复糖损伤PC12细胞中的表达及其对PC12细胞增殖、凋亡的影响

Expression of miR-27a-3p in PC12 cells damaged by oxygen-glucose deprivation and reoxygenation and its effects on proliferation and apoptosis of PC12 cells

目的探讨miR-27a-3p在氧糖缺失-复氧复糖(OGD/R)损伤PC12细胞中的表达及其对OGD/R损伤PC12细胞增殖、凋亡的影响。方法将PC12细胞随机分为正常培养(Normal组)、氧糖缺失2 h后分别复氧复糖3 h、6 h和9 h组,qRT-PCR法检测各时点miR-27a-3p表达。之后将PC12细胞随机分为正常对照(Ctrl组)、氧糖缺失2 h复氧复糖(OGD/R组),慢病毒分别转染miR-27a-3p模拟物、模拟物阴性对照、抑制剂和抑制剂阴性对照入细胞后行OGD/R,分别设为Mimics组、Mimics-Ctrl组、Inhibitor组和Inhibitor-Ctrl组,CCK-8法检测各组细胞复氧复糖后24、48、72和96 h细胞的增殖活性;根据前述结果,选择miR-27a-3p表达差异最大的时间点进行复氧复糖,RT-qPCR法检测各组miR-27a-3p、凋亡相关因子Caspase-3、Bax和Bcl-2 mRNA表达,Western blotting法检测各组Cleaved-Caspase-3、Bax和Bcl-2蛋白表达。结果与Normal组相比,3 h、6 h、9 h组miR-27a-3p表达在OGD/R后均降低(P均<0.05),6 h组呈最低(P<0.05)。与Ctrl组比较,复氧复糖后48、72和96 h,OGD/R组细胞增殖活性均降低(P均<0.05);与Mimics-Ctrl组比较,24、48、72和96 h时Mimics组细胞的增殖活性均升高(P均<0.05);与Inhibitor-Ctrl组比较,72 h和96 h时Inhibitor组细胞增殖活性均降低(P均<0.05)。复氧复糖后6 h,与Ctrl组比较,OGD/R组miR-27a-3p表达降低(P<0.05),Caspase-3、Baxm RNA和蛋白相对表达量均升高(P均<0.05),Bcl-2表达量降低(P<0.05);过表达miR-27a-3p后,miR-27a-3p表达升高(P<0.05),Caspase-3、BaxmRNA和蛋白相对表达量均降低(P均<0.05),Bcl-2表达量升高(P<0.05),抑制miR-27a-3p表达后结果与之相反。结论miR-27a-3p在OGD/R损伤PC12细胞中呈低表达,miR-27a-3p能促进OGD/R损伤PC12细胞的增殖并抑制其凋亡。

Objective To investigate the expression of miR-27a-3p in oxygen/glucose deprivation and reoxygenation(OGD/R)-injured PC12 cells and the effects of miR-27a-3p on the proliferation and apoptosis of OGD/R-injured PC12 cells.Methods PC12 cells were randomly divided into the normal group and reoxygenation groups for 3 h,6 h and 9 h after 2 h of oxygen/glucose deprivation.The expression of miR-27a-3p was detected by qRT-PCR.Then,PC12 cells were randomly divided into the normal control group(Ctrl group),OGD/R group,and OGD/R was performed after transfection with lentivirus carrying miR-27a-3p mimics,mimics negative control,inhibitors and inhibitors negative control,which were taken as the Mimics group,Mimics-Ctrl group,Inhibitor group,and Inhibitor-Ctrl group.The proliferation activity of reoxygenation for 24,48,72 and 96 h was assessed by CCK-8 assay.The mRNA expression levels of miR-27a-3p,apopto‐sis-related factors Caspase-3,Bax and Bcl-2 were detected by RT-qPCR,and the protein expression levels of Cleaved-Cas‐pase-3,Bax,and Bcl-2 were detected by Western blotting.Results The expression of miR-27a-3p decreased in the 3 h,6 h,and 9 h groups after OGD/R as compared with that of the Normal group(P<0.05),with the lowest expression in the 6 h group(P<0.05).Compared with the Ctrl group,the proliferation activity of cells in the OGD/R group decreased at 48,72 and 96 h after reoxygenation(P<0.05).Compared with the Mimics-Ctrl group,the proliferative activity of cells in the Mimics group increased at 24,48,72 and 96 h(P<0.05).The proliferative activity of cells in the Inhibitor group de-creased at 72 and 96 h as compared with that of the Inhibitor-Ctrl group(P<0.05).Moreover,compared with the Ctrl group,the expression of miR-27a-3p in the OGD/R group decreased(P<0.05),the mRNA and protein expression levels of Caspase-3 and Bax increased(both P<0.05),and the expression of Bcl-2 decreased(P<0.05).However,miR-27a-3p overexpression suppressed the mRNA and protein expression levels of Caspase-3 and Bax(both P<0.05)and increased the expression of Bcl-2(P<0.05),but miR-27a-3p inhibitor reversed these results.Conclusion The expression of miR-27a-3p is low in OGD/R-injured PC12 cells,and miR-27a-3p could promote the proliferation and inhibit the apoptosis of OGD/R-injured PC12 cells.