miR-16-5p、IRAK2基因对关节炎软骨细胞增殖、凋亡的影响及其机制  

Effects of miR-16-5p and IRAK2 genes on proliferation and apoptosis of arthritic chondrocytes

作　　者：于云祥 龚泰芳[1] 刘小涛[1] 柯文[1] 李彬彬[1] YU Yunxiang;GONG Taifang;LIU Xiaotao;KE Wen;LI Binbin(Taihe Hospital of Shiyan City,Hubei 442000,China)

机构地区：[1]湖北省十堰市太和医院

出　　处：《山东医药》2019年第36期37-41,共5页Shandong Medical Journal

摘　　要：目的观察miR-16-5p、IRAK2基因对脂多糖(LPS)诱导的胎鼠骨关节炎软骨细胞增殖、凋亡的影响,并探讨其机制。方法剪取胎鼠膝关节获取软骨细胞,常规传代培养。通过靶基因预测网站starBase预测miR-16-5p靶基因,采用荧光素酶报告基因实验进行验证。取对数生长期软骨细胞分成8组。LPS组加入LPS诱导骨关节炎软骨细胞模型,NC组加入等量培养基;LPS+miR-16-5p组转染miR-16-5p模拟物(miR-16-5p mimics)后加入LPS,LPS+miR-con组转染对照模拟物(miR-con)后加入LPS;LPS+si-IRAK2组转染IRAK2 siRNA(si-IRAK2)后加入LPS,LPS+si-con组转染对照siRNA(si-con)后加入LPS;LPS+miR-16-5p+pcDNA-IRAK2组转染miR-16-5p mimics、IRAK2过表达质粒(pcDNA-IRAK2)后加入LPS,LPS+miR-16-5p+pcDNA-con组转染miR-16-5p mimics、对照质粒(pcDNA-con)后加入LPS。采用qRT-PCR法检测NC组、LPS组细胞中miR-16-5p、IRAK2 mRNA;采用MTT法测算各组细胞增殖能力(以细胞存活率表示细胞增殖能力);采用流式细胞仪检测各组细胞凋亡情况,计算细胞凋亡率;采用Western Blotting法检测各组细胞中IRAK2、细胞周期蛋白1(CyclinD1)、B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白。结果LPS组细胞中miR-16-5p、IRAK2 mRNA的相对表达量分别为1.00±0.10、4.01±0.41,NC组分别为5.33±0.53、0.85±0.09,两组相比,P均<0.05。LPS组、NC组细胞存活率分别为63.03%±6.31%、100.00%±10.26%,LPS+miR-16-5p组、LPS+miR-con组分别为93.16%±9.32%、65.46%±6.53%,LPS+si-IRAK2组、LPS+si-con组分别为91.46%±9.15%、60.45%±6.05%,LPS+miR-16-5p+pcDNA-IRAK2组、LPS+miR-16-5p+pcDNA-con组分别为68.08%±6.81%、93.16%±9.31%,两组间相比,P均<0.05。LPS组、NC组细胞凋亡率分别为28.46%±2.85%、1.59%±0.16%,LPS+miR-16-5p组、LPS+miR-con组分别为5.09%±0.51%、30.01%±3.02%,LPS+si-IRAK2组、LPS+si-con组分别为4.88%±0.50%、29.10%±2.91%,LPS+miR-16-5p+pcDNA-IRAK2组、LPS+miR-16-5p+pcDNA-con组分别为20.89%±2.10%、6.15%±0.62%,两组�Objective To observe the effects of miR-16-5p and IRAK2 genes on the proliferation and apoptosis of fetal rat osteoarthritis chondrocytes induced by lipopolysaccharide(LPS),and to explore their mechanisms.Methods Chondrocytes were harvested from fetal rat knee joints and routinely subcultured.The miR-16-5p target gene was predicted by the target gene prediction website starBase,and verified by luciferase reporter gene experiments.Chondrocytes in logarithmic growth phase were divided into eight groups.The LPS group was added with LPS-induced osteoarthritis chondrocyte model,and the NC group was added with equal amount of medium.The LPS+miR-16-5p group was transfected with miR-16-5p mimics(miR-16-5p mimics)and then added with LPS.The LPS+miR-con group was transfected with control mimics(miR-con)and then added with LPS.The LPS+si-IRAK2 group was transfected with IRAK2 siRNA(si-IRAK2)and added with LPS.The LPS+si-con group was transfected with control siRNA(si-con)and added with LPS.LPS+miR-16-5p+pcDNA-IRAK2 group was transfected with miR-16-5p mimics,IRAK2 overexpression plasmid(pcDNA-IRAK2),and was added with LPS.LPS+miR-16-5p+pcDNA-con group was transfected with miR-16-5p mimics and control plasmid(pcDNA-con)and were added with LPS.QRT-PCR was used to detect the miR-16-5p and IRAK2 mRNA in NC and LPS cells.The cell survival rate of each group was measured by the MTT(the cell proliferation ability was expressed by the cell survival rate).Flow cytometry was used to detect the apoptosis of each group,and the apoptosis rate was calculated.Western blotting was used to detect IRAK2,CyclinD1,Bax,Bcl-2 proteins in each group.Results The relative expression levels of miR-16-5p and IRAK2 mRNA in the LPS group were 1.00±0.10 and 4.01±0.41,and those in the NC group were 5.33±0.53 and 0.85±0.09,respectively,with statistically significant difference(P<0.05).The cell survival rates of the LPS group and NC group were 63.03%±6.31%and 100.00%±10.26%,and those in the LPS+miR-16-5p group and LPS+miR-con group were 93.16%±9.32%

关 键 词：微小RNA 微小RNA-16-5p IRAK2基因 骨关节炎 软骨细胞 细胞增殖 细胞凋亡 细胞实验 

分 类 号：R684.3[医药卫生—骨科学]