摘要
目的探讨miR-26a对类风湿关节炎滑膜成纤维细胞增殖及迁移的影响及分子机制。方法实验设置对照(Con)组、脂多糖(LPS)组、LPS+miR-NC组、LPS+miR-26a组、LPS+pcDNA3.1组、LPS+pcDNA3.1-ADAM10组、LPS+miR-26a+pcDNA3.1组、LPS+miR-26a+pcDNA3.1-ADAM10组。实时荧光定量PCR(RT-qPCR)检测miR-26a表达水平;细胞计数试剂盒8(CCK-8)检测细胞活性;克隆形成实验检测细胞克隆形成数;蛋白质印迹(Western blot)法检测解整合素金属蛋白酶10(ADAM10)、细胞增殖核抗原Ki67(Ki-67)、神经钙黏蛋白(N-cadherin)、上皮钙黏蛋白(E-cadherin)蛋白表达;Transwell检测细胞迁移;双荧光素酶报告实验验证miR-26a和ADAM10的靶向关系。结果与对照组相比,LPS组miR-26a表达水平显著降低,细胞活性显著升高,克隆形成数及迁移细胞数显著升高,Ki67、N-cadherin表达水平显著升高,E-cadherin表达水平显著降低(P<0.05)。过表达miR-26a抑制类风湿关节炎滑膜成纤维细胞增殖、迁移。miR-26a靶向ADAM10,过表达ADAM10逆转了miR-26a对类风湿关节炎滑膜成纤维细胞增殖、迁移的抑制作用。结论过表达miR-26a可能通过靶向下调ADAM10抑制类风湿关节炎滑膜成纤维细胞的增殖、迁移。
Objective To investigate the effect and mechanism of miR-26a on the proliferation and migration of rheumatoid arthritis synovial fibroblasts.Methods Experimental settings:control(Con)group,lipopolysaccharide(LPS)group,LPS+miR-NC group,LPS+miR-26a group,LPS+pcDNA3.1 group,LPS+pcDNA3.1-ADAM10 group,LPS+miR-26a+pcDNA3.1 group,LPS+miR-26a+pcDNA3.1-ADAM10 group.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect miR-26a expression.Cell Count-ing Kit 8(CCK-8)was used to detect cell viability;clone formation test was used to detect the number of cell clone formation.Western blot was used to detect the expression of a disintegrin and metalloproteinase 10(AD-AM10),cell proliferation nuclear antigen Ki67(Ki-67),neurocadherin(N-cadherin),and epithelial cad-herin(E-cadherin)protein expression.Transwell was used to detect cell migration;dual luciferase reporting experiment was used to verify the targeting relationship between miR-26a and ADAM10.Results Compared with the control group,the expression of miR-26a in the LPS group was significantly reduced,the cell activi-ty,the number of colony formation,the number of migrating cells,the expressions of Ki67 and N-cadherin were significantly increased,and the expression of E-cadherin was significantly decreased(P<0.05).Overex-pression of miR-26a can inhibit the proliferation and migration of rheumatoid arthritis synovial fibroblasts.miR-26a targets ADAM10,overexpression of ADAM10 can reverse the inhibitory effect of miR-26a on the prolifer-ation and migration of rheumatoid arthritis synovial fibroblasts.Conclusions Overexpression of miR-26a may inhibit the proliferation and migration of rheumatoid arthritis synovial fibroblasts by targeting down-regula-tion of ADAM10.
作者
于守杰
张振春
厉彦山
张丽丽
YU Shoujie;ZHANG Zhenchun;LI Yanshan;ZHANG Lili(Department of rheumatology,linyi people's hospital,Linyi,Shangdong,China,276000)
出处
《分子诊断与治疗杂志》
2020年第6期808-812,共5页
Journal of Molecular Diagnostics and Therapy
基金
临沂市科技发展项目(201515002)。
