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目的:探究miR-199a通过靶向调节ATP13A2对6-OHDA诱导的PC12细胞氧化应激的影响。方法:收集正常大鼠和帕金森(PD)模型大鼠脑组织,培养6-OHDA诱导的PC12细胞,利用Real-time PCR检测miR-199a和ATP13A2的表达,Western Blot检测ATP13A2表达。分别转染miR-199a类似物和miR-199a抑制剂,利用ELISA检测ROS、MDA、SOD和GSH-Px的表达。双荧光素酶报告基因检测miR-199a与ATP13A2的靶向关系。Western Blot法检测ATP13A2和JNK/c-Jun表达的影响。MTT法检测细胞增殖的影响。结果:PD脑组织和细胞中miR-199a表达显著降低,ATP13A2表达显著升高(P<0.01)。过表达miR-199a可显著降低6-OHDA诱导的PC12细胞的ROS、MDA活性,升高SOD、GSH-Px活性(P<0.01)。miR-199a过表达显著降低荧光素酶报告基因的荧光强度,而结合位点突变后荧光素酶活性不变(P<0.01)。过表达miR-199a显著降低ATP13A2的表达并抑制JNK和c-Jun的磷酸化水平,茴香霉素则升高ATP13A2表达并激活JNK和c-Jun的磷酸化水平,过表达miR-199a且给予茴香霉素处理可使JNK和c-Jun的磷酸化水平恢复到6-OHDA-PC12组水平(P<0.01)。过表达miR-199a抑制6-OHDA诱导的PC12细胞增殖,茴香霉素则促进其增殖,过表达miR-199a且给予茴香霉素处理可使细胞增殖率恢复到6-OHDA-PC12组水平(P<0.01)。结论:miR-199a可通过靶向调节ATP13A2抑制6-OHDA诱导的PC12细胞氧化应激并抑制JNK/c-Jun活化。
AIM: To investigate the effect of miR-199a on 6-OHDA-induced oxidative stress in PC12 cells by targeting ATP13A2. Methods: The PC12 cells induced by 6-OHDA were cultured in normal rats and Parkinson’s model rats. The expression of miR-199a and ATP13A2 was detected by Real-time PCR and the expression of ATP13A2 by Western Blot. MiR-199a and miR-199a were transfected respectively, and the expressions of ROS, MDA, SOD and GSH-Px were detected by ELISA. Dual luciferase reporter gene detection of miR-199a and ATP13A2 targeting relationship. Western Blot assay ATP13A2 and JNK / c-Jun expression. The effects of MTT assay on cell proliferation. Results: The expression of miR-199a in PD brain tissue and cells was significantly decreased and the expression of ATP13A2 was significantly increased (P <0.01). Overexpression of miR-199a significantly decreased 6-OHDA-induced ROS and MDA activities and increased activities of SOD and GSH-Px in PC12 cells (P <0.01). Overexpression of miR-199a significantly reduced the fluorescence intensity of luciferase reporter gene, while the activity of luciferase did not change (P <0.01). Overexpression of miR-199a significantly reduced the expression of ATP13A2 and inhibited the phosphorylation of JNK and c-Jun, whereas anisomycin increased the expression of ATP13A2 and activated the phosphorylation of JNK and c-Jun, overexpression of miR-199a and administration of fennel Renalmycin treatment restored the phosphorylation levels of JNK and c-Jun to the 6-OHDA-PC12 group (P <0.01). Overexpression of miR-199a inhibited the proliferation of PC12 cells induced by 6-OHDA, whereas the administration of anisomycin promoted the proliferation of PC12 cells. Overexpression of miR-199a and administration of anisomycin restored the proliferation of PC12 cells to 6-OHDA-PC12 group (P <0.01). Conclusion: miR-199a can regulate ATP13A2-induced 6-OHDA-induced oxidative stress in PC12 cells and inhibit JNK / c-Jun activation.