摘要
目的探讨miR-193a-3p对氧化型低密度脂蛋白(oxLDL)诱导的血管内皮细胞损伤的影响及其作用机制。方法体外培养人脐静脉血管内皮细胞株(EVC-304),使用oxLDL处理EVC-304细胞。采用qRT-PCR与Western blot分别检测miR-193a-3p、S100钙结合蛋白A4(S100A4)的表达水平。分别将miR-NC、miR-193a-3p mimics、si-NC、si-S100A4、miR-193a-3p mimics与pcDNA、miR-193a-3p mimics与pcDNA-S100A4转染至oxLDL诱导的EVC-304细胞。采用硫代巴比妥酸法检测MDA含量,采用黄嘌呤氧化酶法检测SOD活性,检测GSH-Px活性;流式细胞术检测各组细胞凋亡率;双荧光素酶报告实验验证miR-193a-3p与S100A4的靶向关系;Western blot检测细胞中B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达量。结果OxLDL处理后miR-193a-3p的表达水平降低(P<0.05),S100A4的表达水平升高(P<0.05),MDA的含量升高(P<0.05),SOD、GSH-Px的活性降低(P<0.05),Bcl-2蛋白水平降低(P<0.05),细胞凋亡率升高(P<0.05),Bax蛋白水平升高(P<0.05);转染miR-193a-3p mimics后可降低MDA含量、细胞凋亡率及Bax蛋白水平(P<0.05),提高SOD、GSH-Px活性及Bcl-2蛋白水平(P<0.05),而转染si-S100A4与其作用相似;双荧光素酶报告实验证实miR-193a-3p能够靶向结合S100A4;S100A4过表达能够明显减弱miR-193a-3p过表达对oxLDL诱导的血管内皮细胞损伤的作用。结论MiR-193a-3p可能通过靶向调控S100A4表达而减轻oxLDL诱导的血管内皮细胞氧化损伤,并抑制细胞凋亡。
Objective To discuss the influence of miR-193a-3p on vascular endothelial cell injury induced by oxidized low density lipoprotein(oxLDL)and mechanism.Methods Human umbilical vein endothelial cell strain(EVC-304)was cultured in vitro and treated with oxLDL.The expressions of miR-193a-3p and S100 calcium-binding protein A4(S100A4)were detected by using qRT-PCR and Western blotting assay respectively.MiR-NC,miR-193a-3p mimics,si-NC,si-S100A4,miR-193a-3p mimics,pcDNA,miR-193a-3p mimics and pcDNA-S100A4 were transfected into oxLDL-induced EVC-304.The content of malondialdehyde(MDA)was detected by using thiobarbituric acid method,activity of superoxide dismutase(SOD)was detected by using xanthine oxidation method,and activity of glutathione peroxidease(GSH-PX)was detected by using cell counting kit.The apoptotic rate in different groups was detected by using flow cytometry,and targeting relationship between miR-193a-3p and S100A4 was verified by using dual-luciferase reporter gene assay.The expressions of Bcl-2-associated X protein(Bax)and B-cell lymphoma-2(Bcl-2)were detected by using Western blotting assay.Results After oxLDL treatment,miR-193a-3p expression decreased(P<0.05),S100A4 expression increased(P<0.05),MDA content increased(P<0.05),activities of SOD and GSH-Px decreased(P<0.05),Bcl-2 level decreased(P<0.05),apoptotic rate increased(P<0.05),and Bax level increased(P<0.05).After miR-193a-3p mimics transfection,MDA content,apoptotic rate and Bax level decreased(P<0.05),activities of SOD and GSH-Px and Bcl-2 level increased(P<0.05),and the effect of si-S100A4 transfection was similar to that of miR-193a-3p mimics transfection.The results of dual-luciferase reporter gene assay verified that miR-193a-3p was able to target to combine S100A4.The over-expression of S100A4 was able to reduce significantly the effect of miR-193a-3p over-expression on vascular endothelial cell injury induced by oxLDL.Conclusion MiR-193a-3p can relieve oxLDL-reduced oxidative damage of vascular endothelial cells and inhibit apoptosis through target regulation of S100A4 expression.
作者
王彬
齐县伟
张宪亮
Wang Bin;Qi Xianwei;Zhang Xianliang(Department of Intervention Therapy,Central Hospital of Zhoukou City,Zhoukou 466000,China;不详)
出处
《中国循证心血管医学杂志》
2021年第6期742-746,751,共6页
Chinese Journal of Evidence-Based Cardiovascular Medicine
