miRNA-30b-3p靶向ZNRF1调控草酸钠诱导的HK-2细胞凋亡和氧化应激的研究  

miR-30b-3p Regulates Sodium Oxalate Induced Apoptosis and Oxidative Stress in HK-2 Cells by Targeting ZNRF1

作　　者：于大鹏[1] 丁友鹏 李勉洲 张荣远[1,2] YU Dapeng;DING Youpeng;LI Mianzhou;ZHANG Rongyuan(Department of Urology,Jining First People’s Hospital,Jining,Shandong,272100,China;Shenzhen Research Institute,the Chinese University of Hong Kong,Shenzhen,Guangdong,518172,China)

机构地区：[1]济宁市第一人民医院泌尿外科,山东省济宁市272100 [2]香港中文大学深圳研究院,广东省深圳市518172

出　　处：《医学分子生物学杂志》2024年第1期9-16,共8页Journal of Medical Molecular Biology

基　　金：山东省优秀中青年科学家科研奖励基金(No.BS2018SW115)。

摘　　要：目的探索miRNA-30b-3p靶向ZNRF1调控草酸钠诱导的HK-2细胞凋亡和氧化应激的研究并探讨其作用机制。方法将0.2、0.6μmol/L的草酸钠处理HK-2细胞,miR-30b-3p mimic和miR-30b-3p inhibitor及其相对应的对照物转染到HK-2细胞,CCK-8、Transwell和划痕实验分别检测HK-2细胞的增殖、侵袭和迁移能力;流式细胞术测量细胞内ROS,ELISA法检测LDH、MDA、GSH活性,蛋白质印迹分析Bax、Bcl-2的表达情况;荧光素酶报告基因检测验证miRNA-30b-3p及ZNRF1之间的靶向关系;RNA免疫沉淀分析miRNA-30b-3p及ZNRF1之间的作用关系。结果草酸钠处理HK-2显著增加ROS,LDH和MDA水平,GSH含量明显降低(P<0.01),抑制HK-2细胞的增殖、迁移和侵袭,促进其凋亡,与草酸钠的浓度呈现剂量依赖关系,miR-30b-3p促进草酸钠诱导对HK-2细胞的影响;靶标预测和荧光素酶测定表明ZNRF1是HK-2细胞中miR-30b-3p的直接靶标,且沉默ZNRF1逆转miR-30b-3p对草酸钠诱导的HK-2细胞损伤的影响。结论miRNA-30b-3p靶向ZNRF1促进草酸钠诱导的HK-2细胞凋亡和氧化应激,抑制其增殖、侵袭和迁移。Objective To explore the effect of miR-30b-3p on HK-2 cell proliferation,apoptosis and oxidative stress induced by sodium oxalate and the mechanism by targeting ZNRF1.Methods HK-2 cells were treated with 0.2μmol/L or 0.6μmol/L sodium oxalate.miR-30b-3p mimic and miR-30b-3p inhibitor and their corresponding control were transfected into HK-2 cells.The proliferation,invasion and migration of HK-2 cells were detected by CCK-8,transwell and wound-healing assay,respectively.Intracellular ROS was measured by flow cytometry.The activities of LDH,MDA and GSH were detected by ELISA.The expression levels of Bax and Bcl-2 were analyzed by Western blotting.Luciferase gene reporter assay was used to verify the targeting relationship between miR-30b-3p and ZNRF1.The relationship between miR-30b-3p and ZNRF1 was analyzed by RNA immunoprecipitation.Results Sodium oxalate treatment significantly increased ROS,LDH and MDA levels and decreased GSH levels in HK-2 cells(P<0.01).Sodium oxalate also inhibited the proliferation,migration and invasion,and promoted apoptosis of HK-2 cells.miR-30b-3p promoted the effect of sodium oxalate on HK-2 cells.The results of gene target prediction and luciferase assay indicated that ZNRF1 is a direct target of miR-30b-3p in HK-2 cells,and ZNRF1 silencing reversed the effect of miR-30b-3p on sodium oxalate induced HK-2 cell damage.Conclusion miR-30b-3p can promote the apoptosis and oxidative stress induced by sodium oxalate in HK-2 cells,and inhibit cell proliferation,invasion and migrationvia ZNRF1.

关 键 词：miRNA-30b-3p ZNRF1 草酸钠 HK-2 氧化应激 

分 类 号：R33[医药卫生—人体生理学]