miR-21-5p靶向调控Skp2促进骨关节炎软骨细胞凋亡并抑制自噬  

miR⁃21⁃5p targeted regulation of Skp2 promotes apoptosis and inhibits autophagy in osteoarthritis chondrocytes

作　　者：袁长深[1] 李彦宏 刘晋邑 袁景钊 梅其杰[1] 徐文飞 郭锦荣[1] 段戡[1] 莫坚[3] YUAN Changshen;LI Yanhong;LIU Jinyi;YUAN Jingzhao;MEI Qijie;XU Wenfei;GUO Jinrong;DUAN Kan;MO Jian(Orthopedic Department of the Limbs,The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,China;Graduate School,Guangxi University of Chinese Medicine,Nanning 530000,China;Department of Joint and Sports Medicine,Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011,China)

机构地区：[1]广西中医药大学第一附属医院四肢骨伤科,广西南宁530023 [2]广西中医药大学研究生院,广西南宁530000 [3]广西中医药大学附属瑞康医院关节与运动医学科,广西南宁530011

出　　处：《海南医学院学报》2024年第24期1859-1868,共10页Journal of Hainan Medical University

基　　金：国家自然科学基金资助项目(82060875,82160912);广西自然科学基金(2023GXNSFAA026051);广西中医药大学研究生教育创新计划项目(YCSW2024401)。

摘　　要：目的:从人软骨细胞角度探讨miR-21-5p靶向调控Skp2对OA软骨细胞凋亡、自噬的影响。方法:TargetScan在线网站预测miR-21-5p与Skp2结合位点,双荧光素酶实验验证miR-21-5p与Skp2的直接作用,选择原代人软骨细胞分离与培养,采用CCK-8实验检测细胞增殖能力,qPCR检测miR-21-5p对人软骨细胞中Skp2、Bax、Bcl-2、LC3Ⅰ、LC3Ⅱ、Beclin1、CARM1mRNA表达量,Westernblot检测Skp2、CARM1、Bax、Bcl-2、LC3Ⅱ/Ⅰ、Beclin1蛋白表达水平,后期构建Skp2过表达载体进一步检测相关蛋白表达情况。结果:TargetScan在线网站预测在Skp2 mRNA的3’UTR中有潜在miR-21-5p结合序列;双荧光素酶方法验证miR-21-5p与Skp2存在互作关系;qPCR检测出Skp2过表达验证成功,证实在软骨细胞中Skp2呈低表达;Western blot检测显示miR-21-5p可通过调节Skp2、CARM1、Bax、Bcl-2、LC3Ⅱ/Ⅰ、Beclin1水平,促进细胞凋亡,减少人软骨细胞自噬。结论:miR-21-5p可负向调控Skp2,促进OA软骨细胞凋亡并抑制自噬。Objective:To explore the effect of miR-21-5p targeted regulation of Skp2 on apoptosis and autophagy in OA chondrocytes from the perspective of human chondrocytes.Methods:TargetScan online website predicted the binding site of miR-21-5p and Skp2,the direct interaction between miR-21-5p and Skp2 was verified by dual luciferase assay,primary human chondrocytes were selected for isolation and culture,cell proliferation ability was detected by CCK-8 assay,the effect of miR-21-5p on Skp2 in human chondrocytes,Skp2,Bax,Bcl-2,LC3Ⅰ,LC3Ⅱ,Beclin1,and CARM1 mRNA expression in human chondrocytes was detected by qPCR,and Western blot was used to detect the protein expression level of Skp2,CARM1,Bax,Bcl-2,LC3Ⅱ/Ⅰ,Beclin1,and the construction of Skp2 overexpression vector at a later stage to further detect the expression of related proteins.Results:TargetScan online website predicted that there was a potential miR-21-5p binding sequence in the 3’UTR of Skp2 mRNA;dual-luciferase method verified that there was an interactions between miR-21-5p and Skp2;qPCR detected successful Skp2 overexpression validation,confirming that Skp2 was under-expressed in chondrocytes;Western blot assay showed that miR-21-5p could promote apoptosis and reduce autophagy in human chondrocytes by regulating the levels of Skp2,CARM1,Bax,Bcl-2,LC3Ⅱ/Ⅰ,and Beclin1.Conclusion:miR-21-5p negatively regulates Skp2,promotes apoptosis,and inhibits autophagy in OA chondrocytes.

关 键 词：骨关节炎 软骨细胞 miR-21-5p SKP2 凋亡 自噬 

分 类 号：R684.3[医药卫生—骨科学]