circZNF652靶向miR-496调控糖氧剥夺诱导PC12细胞氧化应激和凋亡的实验研究

Experimental study of circZNF652 regulate oxidative stress and apoptosis of PC12 cells induced by oxgen-glucose deprivation through targeting miR-496

目的探讨circZNF652对糖氧剥夺(OGD)诱导的PC12细胞氧化应激和凋亡的影响及机制。方法体外培养神经细胞PC12,建立OGD模型,检测细胞中circZNF652和miR-496表达量。分别转染si-circZNF652、miR496 mimics或共转染si-circZNF652与anti-miR-496至PC12细胞中,检测乳酸脱氢酶(LDH)、丙二醛(MDA)、细胞凋亡率、细胞中活化的半胱天冬酶(cleaved-caspases)3和cleaved-caspase9蛋白表达。双荧光素酶报告基因实验验证circZNF652和miR-496的调控关系。结果OGD组PC12细胞中circZNF652表达明显高于Con组(t=22.57,P<0.05),miR-496的表达量明显低于Con组(t=40.50,P<0.05);与Con组比较,OGD组PC12细胞培养上清液中LDH释放量、细胞中MDA含量均升高(t分别=20.66、23.88,P均<0.05)。与OGD+si-NC组比较,OGD+si-circZNF652组PC12细胞培养上清液中LDH释放量、细胞中MDA含量均降低(t分别=17.04、20.30,P均<0.05);与Con组比较,OGD组PC12细胞凋亡率、cleaved-caspase3、cleaved-caspase9蛋白表达均升高(t分别=23.95、26.74、28.17,P均<0.05);与OGD+si-NC组比较,OGD+si-circZNF652组PC12细胞凋亡率、cleaved-caspase3、cleaved-caspase9蛋白表达均降低(t分别=20.36、21.61、20.61,P均<0.05)。与OGD+miR-NC组比较,OGD+miR-496组miR-496的表达量明显升高(t=20.22,P<0.05);与OGD+si-circZNF652+anti-miR-NC组比较,OGD+si-circZNF652+anti-miR-496组miR496的表达量明显降低(t=72.00,P<0.05)。结论下调circZNF652可能通过靶向上调miR-496抑制OGD诱导的PC12细胞氧化应激和凋亡。

Objective To investigate the effect and mechanism of circZNF652 in oxygen-glucose deprivation(OGD)-induced oxidative stress and apoptosis of PC12 cells.Methods PC12 cells were cultured in vitro,and the OGD model was established.The expression of circZNF652 and miR-496 in cells was detected.The PC12 cells were trans⁃fected with si-circZNF652,miR-496 mimics,or co-transfected with si-circZNF652 and anti-miR-496,the lactate dehy⁃drogenase(LDH)and malondialdehyde(MDA),cell apoptosis rate,activated caspases 3 and cleaved caspases 9 were detected.The dual luciferase reporter gene experiment was conducted to verify the regulatory relationship between circ⁃ZNF652 and miR-496.Results The expression of circZNF652 in PC12 cells of OGD group was significantly higher than that of Con group(t=22.57,P<0.05),and the expression of miR-496 was significantly lower than that of Con group(t=40.50,P<0.05).Compared with Con group,the level of LDH and the content of MDA in PC12 cell culture su⁃pernatant in OGD group increased(t=20.66,23.88,P<0.05).Compared with OGD+si-NC group,LDH level and MDA content in PC12 cell culture supernatant of OGD+si-circZNF652 group decreased(t=17.04,20.30,P<0.05).Compared with Con group,the apoptosis rate of PC12 cells,the expression of cleaved caspase 3 and cleaved caspase 9 protein in OGD group increased(t=23.95,26.74,28.17,P<0.05).Compared with OGD+si-NC group,the apoptosis rate of PC12 cells and the expression of cleaved caspase 3 and cleaved caspase 9 protein in OGD+si-circZNF652 group decreased(t=20.36,21.61,20.61,P<0.05).Compared with OGD+miR-NC group,the expression of miR-496 in OGD+miR-496 group increased significantly(t=20.22,P<0.05),compared with OGD+si-circZNF652+anti-miR-NC group,the expression of miR-496 in OGD+si-circ⁃ZNF652+anti-miR-496 group decreased significantly(t=72.00,P<0.05).Conclusion Down-regulation of circZNF652 may inhibit OGD-induced oxidative stress and apoptosis of PC12 cells by up-regulation of miR-496.