微小RNA-215-5p/锌指结构E-box同源结合框2促进白细胞介素-1β诱导软骨细胞凋亡在骨关节炎中的作用机制  

MicroRNA-215-5p promotes interleukin-1β-induced chondrocyte apoptosis by targeting zinc finger E-box binding homeobox 2

作　　者：王欢 徐剑 邢丹谋[1] 任东 冯伟 陈焱 张明 吴其鹏 Wang Huan;Xu Jian;Xing Danmou;Ren Dong;Feng Wei;Chen Yan;Zhang Ming;Wu Qipeng(Department of Hand Surgery,Wuhan Fourth Hospital,Wuhan 430030,China;Department of Orthopedics for Children,Wuhan Fourth Hospital,Wuhan 430030,China)

机构地区：[1]武汉市第四医院手外一科,武汉430030 [2]武汉市第四医院小儿骨科,武汉430030

出　　处：《中华实验外科杂志》2023年第1期98-101,共4页Chinese Journal of Experimental Surgery

基　　金：武汉市医学科研项目(WX19Y19)。

摘　　要：目的探讨微小RNA(miR)-215-5p对白细胞介素(IL)-1β诱导的骨关节软骨细胞凋亡的影响及其机制。方法将骨关节炎软骨细胞ATDC5随机分为磷酸盐缓冲液(PBS)处理对照组和IL-1β处理模型组。为了分析miR-215-5p对IL-1β诱导细胞凋亡的影响,进一步的将IL-1β组分为:模型组+miR对照组,模型组+miR-215-5p抑制剂组。细胞转染后采用10 ng/ml IL-1β处理软骨细胞。采用流式细胞术检测细胞凋亡水平。采用实时定量反转录聚合酶链反应(RT-qPCR)检测miR-215-5p和锌指结构E-box同源结合框2(ZEB2)的表达水平。蛋白质印迹法(Western blot)检测ZEB2、p65、磷酸化p65、裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)蛋白水平。将野生型ZEB2的3’-非编码区(pmiR-ZEB2-WT)或者突变型ZEB2的3’-非编码区(pmiR-ZEB2-Mut)分别与miR-215-5p模拟物(miR-215-5p mimic)和对照物(miR-NC)共转染细胞,双荧光素酶报告基因测定实验研究miR-215-5p与ZEB2的相互作用。结果与对照组比较,miR-215-5p在IL-1β诱导软骨细胞凋亡模型中表达上调(2.87±0.07)倍。双荧光素酶报告实验发现ZEB2是miR-215-5p的直接作用靶点。miR-215-5p mimic处理可使转染pmiR-ZEB2-WT的细胞荧光素酶活性降低为miRNA对照组的40.2%,而在转染pmiR-ZEB2-Mut的细胞中miR-215-5p mimic组与对照组比较,差异无统计学意义。miR-215-5p抑制剂使IL-1β诱导的软骨细胞凋亡水平降低为对照组的50.9%,并且可降低软骨细胞凋亡标志物cleaved Caspase-3水平和核因子-κB(NF-κB)p65的磷酸化水平为对照组的68.2%和75.4%。结论miR-215-5p通过靶向ZEB2促进白细胞介素-1β诱导软骨细胞凋亡。Objective To investigate the effect and mechanism of microRNA(miR)-215-5p on interleukin(IL)-1β-induced apoptosis of chondrocytes.Methods Human chondrocyte cells ATDC5 were randomly divided into PBS group and IL-1β group.In order to analyze the effect of miR-215-5p on IL-1β-induced apoptosis,the IL-1β group was further divided into IL-1β+miR control group and IL-1β+miR-215-5p inhibitor group.After miRNA transfection,chondrocytes were treated with 10 ng/ml IL-1β.Cell apoptosis was detected by flow cytometry.The expression levels of miR-215-5p and zinc finger E-box binding homeobox 2(ZEB2)were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The levels of ZEB2,p65,phosphorylated p65 and cleaved cysteinyl aspartate-specific protease-3(cleaved Caspase-3)were examined by Western blotting.The 3′-non-coding region of wild-type ZEB2(pmiR-ZEB2-WT)or the 3′-non-coding region of mutant ZEB2(pmiR-ZEB2-Mut)was co-transfected with miR-215-5p mimic and negative control(miR-NC),respectively.The direct interaction between miR-215-5p and ZEB2 was analyzed by dual luciferase reporter assay.Results Compared with control group,the expression of miR-215-5p was up-regulated by 2.87±0.07 times in IL-1β-induced chondrocyte apoptosis group.Dual luciferase reporter assay indicated that ZEB2 was the direct target of miR-215-5p.The luciferase activity of ATDC5 cells in miR-215-5p mimic group was reduced to 40.2% of that in miR-NC group,but there was no significant difference between the miR-215-5p mimic group and miR-NC group in the cells transfected with pmiR-ZEB2-Mut.The miR-215-5p inhibitor reduced the level of IL-1β-induced chondrocyte apoptosis to 50.9%of the control group,and reduced the levels of cleaved Caspase-3 and nuclear factor-κB(NF-κB)p65 phosphorylation to 68.2% and 75.4% of the control group,respectively.Conclusion This study suggested that microRNA-215-5p may promote IL-1β-induced chondrocyte apoptosis by targeting ZEB2.

关 键 词：骨关节炎 微小RNA 软骨细胞 凋亡 

分 类 号：R684.3[医药卫生—骨科学]