miR-96-5p靶向IRS1对麦芽酚铝致PC12细胞凋亡的影响

Effect of miR-96-5p targeting IRS1 on apoptosis of PC12 cells induced by aluminum maltol

目的探讨miR-96-5p在麦芽酚铝致PC12细胞凋亡中的影响及其作用机制。方法于2021年1月, 取对数生长期的PC12细胞, 分为空白对照组和低、中、高剂量组, 分别采用0、100、200、400 μmol/L的麦芽酚铝处理24 h, 收集各组细胞, 采用流式细胞术检测细胞凋亡率, qRT-PCR检测miR-96-5p和胰岛素受体底物1(IRS1)mRNA表达水平, Western blotting检测半胱氨酸蛋白酶3(Caspase3)、活化型半胱氨酸蛋白酶3(Cleaved-caspase3)、IRS1、磷酸化蛋白激酶B(p-AKT)、磷酸化葡萄糖合成激酶3β(p-GSK3β)蛋白表达水平。双荧光素酶报告基因实验检测miR-96-5p和IRS1的靶向结合关系。采用miR-96-5p inhibitor转染PC12细胞24 h构建miR-96-5p抑制细胞和阴性对照细胞, 将细胞分为空白对照组、阴性对照组、铝染毒组、铝染毒+阴性对照组、铝染毒+miR-96-5p抑制组、miR-96-5p抑制组, 采用miR-96-5p inhibitor和IRS1 siRNA转染PC12细胞24 h后将细胞分为铝染毒+miR-96-5p抑制+阴性对照组和铝染毒+miR-96-5p抑制+IRS1抑制组, 对照组细胞用完全培养基培养, 染毒组细胞采用200 μmol/L麦芽酚铝处理24 h, 收集各组细胞, 检测细胞凋亡率、miR-96-5p和IRS1 mRNA表达水平以及Caspase3、Cleaved-caspase3、IRS1、p-AKT、p-GSK3β蛋白表达水平。结果染毒24 h后, 与空白对照组和低剂量组比较, 中、高剂量组PC12细胞的凋亡率、Caspase3和Cleaved-caspase3蛋白相对表达量, 以及miR-96-5p相对表达量均明显升高, IRS1 mRNA相对表达量明显下降, IRS1、p-AKT和p-GSK3β蛋白相对表达量明显下降(P<0.05)。Targetscan预测和双荧光素酶报告实验均证明IRS1是miR-96-5p的直接靶基因。在转染实验中, 与铝染毒组比较, 铝染毒+miR-96-5p抑制组PC12细胞凋亡率以及Caspase3和Cleaved-caspase3蛋白相对表达量均下降, miR-96-5p的相对表达量明显下降, IRS1 mRNA和IRS1、p-AKT、p-GSK3β蛋白的相对表达量均升高(P<0.05)。在IRS1低表达实验中, 与铝染毒+miR-96-5p抑制+阴性对照组比较, 铝染毒+miR-96-5p抑制+IRS1抑制组中PC12细胞凋亡率、Caspase3和Cleaved-caspase3蛋白相对表达量明显增加, IRS1 mRNA相对表达量以及IRS1、p-AKT和p-GSK3β蛋白相对表达量明显下降(P<0.05)。结论 miR-96-5p表达增加从而靶向抑制IRS1可能是麦芽酚铝染毒导致PC12细胞凋亡的机制之一。

Objective To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum.Methods In January 2021,PC12 cells at logarithmic growth phase were divided into blank control group and low,medium and high dose group.Cells in each group were treated with O,100,200 and 400μmol/L maltol aluminum for 24 hours respectively.Cells were collected and cell apoptosis rates were detected by flow cytometry,miR-96-5p and insulin receptor substrate 1(IRS1)mRNA expressions were detected by qRT-PCR,and the protein expression levels of cysteine protease 3(Caspase3)、activated cysteine protease 3(Cleaved-caspase3)、IRS1、phosphorylated protein kinase B(p-AKT)and phosphorylated glucose synthesis kinase 3β(p-GSK3β)were detected by western blotting.The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment.The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours.The cells were divided into blank control group,negative control group,aluminum exposure group,aluminum exposure+negative control group,aluminum exposure+miR-96-5p inhibition group,and miR-96-5p inhibition group.After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h,the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group.The control group was cultured in complete culture medium,and cells in the aluminum exposure group were treated with 200μmol/L maltol aluminum for 24 hours.Cells in each group were collected and the apoptosis rate,miR-96-5p and IRS1 mRNA expression levels,as well as protein expression levels of Caspase3,Cleaved-caspase3,IRS1,p-AKT,and p-GSK3β were measured.Results After 24 hours of exposure,compared with blank control group and low-dose group,the apoptosis rates,relative expressions of Caspase3 and Cleaved-caspase3 proteins,and relative expressions of miR-96-5p in the medium and high-dose groups ofPC12 cells were significantly increased,while the relative expression levels ofIRSI mRNA,IRS1,AKT and p-GSK3βproteins were significantly decreased(P<0.05).Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p.In the transfection experiment,compared with the aluminum exposure group,the apoptosis rate,the relative expressions of Caspase3 and Cleaved-caspase3 proteins,the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased,while the relative expression levels of IRS1 mRNA and IRS1,p-AKT and p-GSK3βproteins were significantly increased(P<0.05).In the IRS1 low expression experiment,compared with the aluminum exposure+miR-96-5p inhibition+negative control group,the apoptosis rate,the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRSI inhibition group were significantly increased,while the relative expression levels of IRSI mRNA and IRS1,p-AKT and p-GSK3βproteins were significantly decreased(P<0.05).Conclusion The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.