miR-34a通过靶向SIRT1对MPP+诱导的帕金森病模型细胞凋亡和氧化应激损伤的影响

Effect of miR-34a on MPP + induced apoptosis and oxidative stress of Parkinson′s disease model cells by targeting SIRT1 gene

目的:探讨miR-34a通过靶向SIRT1对MPP+诱导的帕金森病模型细胞凋亡和氧化应激损伤的影响。方法:以MPP+处理SH-SY5Y细胞建立帕金森病细胞模型,MTT法检测细胞存活率,RT-PCR检测细胞中miR-34a和SIRT1 mRNA的表达,Western blot检测SIRT1蛋白的表达。脂质体介导转染miR-34a模拟物和miR-34a抑制剂,RT-PCR和Western blot检测miR-34a对SIRT1 mRNA和蛋白表达的影响。采用荧光素酶实验检测miR-34a与SIRT1的靶向关系。将miR-34a抑制剂和SIRT1干扰序列转染至SH-SY5Y细胞后,采用流式细胞仪和LDH、SOD、MDA试剂盒分别检测miR-34a低表达靶向SIRT1对MPP+诱导的SH-SY5Y细胞凋亡和氧化应激损伤的影响。结果:与对照组相比,500μmol/L、1000μmol/L和2000μmol/L MPP+能够降低SH-SY5Y细胞的存活率、上调miR-34a和下调SIRT1表达(P<0.05),且呈浓度依赖性。miR-34a模拟物可引起SIRT1 mRNA和蛋白表达降低,miR-34a抑制剂可引起SIRT1 mRNA和蛋白表达升高;荧光素酶实验证实SIRT1是miR-34a的靶基因。2000μmol/L MPP+可诱导SH-SY5Y细胞凋亡,细胞上清液中MDA含量和LDH活性显著升高,而SOD活性降低;转染miR-34a抑制剂成功下调miR-34a表达后可逆转MPP+引起的上述变化,而转染SIRT1干扰序列成功下调SIRT1表达后可减弱miR-34a低表达引起的上述变化。结论:miR-34a低表达可通过靶向调控SIRT1抑制MPP+诱导的帕金森病模型细胞凋亡和氧化应激损伤。

Objective:To investigate the effects of miR-34a on MPP+induced apoptosis and oxidative stress injury in Parkinson′s disease model by targeting SIRT1.Methods:Kinsen′s disease cell model was established by MPP+treatment of SH-SY5Y cells.MTT assay was used to detect cell viability.RT-PCR was used to detect the expression of miR-34a and SIRT1 in SH-SY5Y cells.Western blot was used to detect the expression of SIRT1 protein.Liposome-mediated transfection of mimics and inhibitors of miR-34a was performed.RT-PCR and Western blot were used to detect the effects of miR-34a on SIRT1 gene and protein expression.Luciferase assay was used to detect the targeting relationship between miR-34a and SIRT1.miR-34a inhibitors and SIRT1 interference sequence were transfected into SH-SY5Y cells.Flow cytometry and LDH,SOD and MDA kits were used to detect the effects of SIRT1 targeting low expression of miR-34a on MPP+induced SH-SY5Y cell apoptosis and oxidative stress injury.Results:Compared with the control group,500,1000 and 2000μmol/L MPP+could decrease the survival rate of SH-SY5Y cells,up-regulate the expression of miR-34a and down-regulate the expression of SIRT1 in a concentration-dependent manner(P<0.05).The expression of SIRT1 was decreased by mimics of miR-34a and increased by inhibitors of miR-34a.Luciferase assay confirmed that SIRT1 was the target gene of miR-34a.2000μmol/L MPP+could induce SH-SY5Y cell apoptosis,and the MDA content and LDH activity in supernatant increased significantly,while the SOD activity decreased.The above changes induced by MPP+could be reversed by the successful down-regulation of the expression of miR-34a by the inhibitor of miR-34a,while the down-regulation of the expression of miR-34a by the SIRT1 interference sequence could attenuate the above changes caused by the down-regulation of the expression of miR-34a.Conclusion:The low expression of miR-34a can inhibit MPP+induced apoptosis and oxidative stress injury in Parkinson′s disease model by targeting SIRT1.