miR-133b靶向抑制SGTB对oxLDL诱导的血管内皮细胞损伤的影响

Effects of miR-133b on oxLDL-induced vascular endothelial cell injury by targeting SGTB

目的:探讨微小RNA-133b(miR-133b)靶向抑制富含谷氨酰胺三十四肽重复序列的小蛋白质分子(SGTB)对氧化低密度脂蛋白(oxLDL)诱导的血管内皮细胞损伤的影响。方法:采用100μg/ml的oxLDL诱导人脐静脉血管内皮细胞(EVC-304)24 h构建血管内皮细胞损伤模型。将EVC-304细胞分为对照组、oxLDL组(oxLDL处理)、oxLDL+miR-NC组(转染20 nmol/L miR-NC+oxLDL处理)、oxLDL+miR-133b组(转染20 nmol/L miR-133b mimics+oxLDL处理)、oxLDL+si-NC组(转染20 nmol/L si-NC+oxLDL处理)、oxLDL+si-SGTB组(转染20 nmol/L si-SGTB+oxLDL处理)、oxLDL+miR-133b+pcDNA组(转染20 nmol/L si-SGTB和pcDNA+oxLDL处理)、oxLDL+miR-133b+pcDNA-SGTB组(转染20 nmol/L si-SGTB和pcDNA-SGTB处理)。实时荧光定量PCR(qRT-PCR)和蛋白质印记(Western blot)检测miR-133b和SGTB的表达水平;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性;Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。双荧光素酶报告基因实验和Western blot验证miR-133b对SGTB的靶向调控关系。结果:与对照组比较,oxLDL诱导后EVC-304细胞miR-133b、Bcl-2的表达水平显著降低(P<0.05),SGTB、Bax的表达水平显著升高(P<0.05),MDA含量和细胞凋亡率显著增加(P<0.05),SOD和GSH-Px活性显著降低(P<0.05)。过表达miR-133b或干扰SGTB均可抑制oxLDL诱导的EVC-304细胞凋亡和氧化应激损伤(P<0.05)。miR-133b与SGTB直接结合,过表达miR-133b显著下调SGTB表达(P<0.05),抑制miR-133b显著上调SGTB表达(P<0.05)。过表达SGTB可逆转过表达miR-133b对oxLDL诱导的血管内皮细胞损伤的影响(P<0.05)。结论:miR-133b通过靶向抑制SGTB的表达,可减轻oxLDL诱导的血管内皮细胞氧化应激损伤和细胞凋亡。

Objective:To investigate the effect of microRNA-133b(miR-133b)on oxidized low density lipoprotein(oxLDL)induced vascular endothelial cell injury by targeting small protein molecules rich in glutamine 34-tetrapeptide repeats(SGTB).Methods:Human umbilical vein endothelial cells(EVC-304)were induced by 100μg/ml oxLDL for 24 h to construct a vascular endothelial cell injury model.EVC-304 cells were divided into control group,oxLDL group(oxLDL treatment),oxLDL+miR-NC group(transfectted with 20 nmol/L miR-NC+oxLDL treatment),oxLDL+miR-133b group(transfectted with 20 nmol/L miR-133b mimics+oxLDL treatment),oxLDL+si-NC group(transfectted with 20 nmol/L si-NC+oxLDL treatment),oxLDL+si-SGTB group(transfected with 20 nmol/L si-SGTB+oxLDL treatment),oxLDL+miR-133b+pcDNA group(transfected with 20 nmol/L si-SGTB and pcDNA+oxLDL),oxLDL+miR-133b+pcDNA-SGTB group(transfected with 20 nmol/L si-SGTB and pcDNA-SGTB),9 wells in each group.Real-time quantitative PCR(qRT-PCR)and Western blot were used to detect the expression levels of miR-133b and SGTB;flow cytometry was used to detect cell apoptosis;kits were used to detect malondialdehyde(MDA)content and the activities of superoxide disproportionation enzyme(SOD)and glutathione peroxidase(GSH-Px).The expression levels of Bcl-2 and Bax protein were detected by Western blot.The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory between miR-133b and SGTB.Results:Compared with the control group,the expressions of miR-133b and Bcl-2 in EVC-304 cells were decreased significantly after oxLDL induction,while the expression levels of SGTB and Bax were sincreased ignificantly(P<0.05),the MDA content and apoptosis rate were increased significantly(P<0.05),and the activities of SOD and GSH-Px were decreased significantly(P<0.05).Over-expression of miR-133b or interfering with SGTB inhibited oxLDL-induced apoptosis and oxidative stress in EVC-304 cells(P<0.05).miR-133b directly bound to SGTB,miR-133b overexpression significantly down-regulated SGTB expression(P<0.05),miR-133b inhibition significantly up-regulated SGTB expression(P<0.05)Over-expression of SGTB reversed the effect of over-expressing miR-133b on oxLDL-induced vascular endothelial cell injury(P<0.05).Conclusion:miR-133b could attenuate oxidative stress damage and apoptosis induced by oxLDL in vascular endothelial cells by targeting and inhibiting SGTB expression.