hsa_circ_0000734吸附miR-16-5p抑制类风湿关节炎滑膜成纤维细胞的生长和迁移

The regulatory effects of hsa_circ_0000734 on the growth and migration of rheumatoid arthritis synovial fibroblasts by adsorbing miR-16-5p

目的探讨hsa_circ_0000734对类风湿关节炎滑膜成纤维细胞MH7A增殖、凋亡和迁移的调控作用及潜在机制。方法体外培养MH7A细胞,使用hsa_circ_0000734过表达质粒转染MH7A细胞,实验分为空白对照组(Mock组)、转染对照组(NC组)和转染组(hsa_circ_0000734组),采用实时荧光定量PCR(qRT-PCR)检测各组细胞中hsa_circ_0000734的表达情况,细胞计数试剂盒-8(CCK-8)检测细胞增殖活力,Annexin V-FITC/PI双染色法检测细胞凋亡率,Transwell实验检测细胞迁移能力,蛋白免疫印迹(Western blot)法分析增殖细胞核抗原(PCNA)、活化的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的表达。双荧光素酶报告基因实验分析hsa_circ_0000734和miR-16-5p之间的靶向关系。通过转染过表达miR-16-5p,采用同样的方法分析细胞增殖、凋亡和迁移能力的变化。结果与Mock组和NC组比较,hsa_circ_0000734组细胞中hsa_circ_0000734和Cleaved Caspase-3的表达及细胞凋亡率均明显升高(P<0.05),而miR-16-5p、PCNA、MMP-2和MMP-9的表达、细胞增殖活力和迁移能力均明显降低(P<0.05);Mock组和NC组比较,各指标差异无统计学意义(P>0.05)。双荧光素酶报告基因实验证实了hsa_circ_0000734和miR-16-5p存在靶向关系。过表达miR-16-5p能够逆转过表达hsa_circ_0000734对MH7A细胞生长和迁移的抑制作用。结论hsa_circ_0000734可通过靶向调控miR-16-5p的表达抑制MH7A细胞的生长和迁移。

Objective To investigate the regulatory effects of hsa_circ_0000734 on the growth and migration of rheumatoid arthritis synovial fibroblasts-MH7A,and to explore potential action mechanism.Methods MH7A cells were cultured in vitro,and hsa_circ_0000734 overexpression plasmid was transfected into MH7A cells.The experiment was divided into blank control(Mock)group,transfection control(NC)group and transfection(hsa_circ_0000734)group.The real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression levels of hsa_circ_0000734;cell counting kit-8(CCK-8)was used to detect cell proliferation;Annexin V-FITC/PI double staining method was used to detect cell apoptosis rate;Transwell test was used to detect cell migration ability,protein immunity;Western Blot was used to detect the expressions of proliferating cell nuclear antigen(PCNA),activated aspartate proteolytic enzyme 3(Cleaved Caspase-3),matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9).The dual luciferase reporter gene experiment was used to analyze the targeting correlation between hsa_circ_0000734 and miR-16-5p.By transfection and overexpression of miR-16-5p,the same methods mentioned above were used to analyze the changes of proliferation,apoptosis and migration ability of the cells.Results Compared with those in Mock group and NC groups,the expression levels of hsa_circ_0000734 and Cleaved Caspase-3 as well as the apoptosis rate in hsa_circ_0000734 group were significantly increased(P<0.05),however,the expression levels of miR-16-5p,PCNA,MMP-2 and MMP-9 as well as the cell proliferation activity and migration ability were significantly decreased(P<0.05),there were no significant differences in above indexes between Mock group and NC group(P>0.05).The dual luciferase reporter gene experiment confirmed that hsa_circ_0000734 and miR-16-5p had a targeting relationship.Moreover overexpression of miR-16-5p could reverse the inhibitory effects of overexpression of hsa_circ_0000734 on the growth and migration of MH7A cells.Conclusion The hsa_circ_0000734 can inhibit the growth and migration of MH7A cells by targetedly regulating the expression of miR-16-5p.