miR-106a-5p通过调控TLR4表达对类风湿关节炎滑膜成纤维细胞增殖和凋亡的影响

Effect of miR-106a-5p on the proliferation and apoptosis of rheumatoid arthritis synovial fi⁃broblasts by regulating the expression of TLR4

目的:探讨miR-106a-5p通过调控TLR4对类风湿关节炎(RA)滑膜成纤维细胞增殖和凋亡的影响。方法:体外培养RA滑膜成纤维细胞MH7A,将细胞分为8组:miR-NC组(转染miR-NC)、miR-106a-5p组(转染miR-106a-5p)、si-NC组(转染siNC)、si-TLR4组(转染si-TLR4)、anti-miR-NC组(转染anti-miR-NC)、anti-miR-106a-5p组(转染anti-miR-106a-5p)、miR-106a-5p+pcDNA组(共转染miR-106a-5p和pcDNA)以及miR-106a-5p+TLR4组(共转染miR-106a-5p和TLR4)。采用qRT-PCR检测RA患者滑膜中miR-106a-5p和TLR4 m RNA的表达;MTT检测细胞增殖情况;流式细胞术检测细胞凋亡情况;Western blotting检测TLR4、PCNA、Bax和Bcl-2蛋白表达;双荧光素酶实验验证miR-106a-5p和TLR4关系。结果:与对照组相比,RA滑膜组织中miR-106a-5p表达明显降低(P<0.05),TLR4 mRNA和蛋白表达明显增加(P<0.05)。与miR-NC组相比,miR-106a-5p组的miR-106a-5p表达、细胞凋亡率和Bax蛋白表达明显增加(P<0.05);48 h及72 h的细胞OD值、PCNA和Bcl-2蛋白表达明显降低(P<0.05)。与si-NC组相比,si-TLR4组的TLR4 mRNA及蛋白表达、48 h及72 h的细胞OD值、PCNA和Bcl-2蛋白表达明显降低(P<0.05);细胞凋亡率和Bax蛋白表达明显增加(P<0.05)。与miR-NC组相比,miR-106a-5p组的TLR4-WT荧光素酶活性明显降低(P<0.05),而TLR4-MUT荧光素酶素酶活性无明显变化。与anti-miR-NC组相比,anti-miR-106a-5p组中miR-106a-5p表达明显降低(P<0.05),TLR4蛋白表达明显增加(P<0.05)。与miR-106a-5p+pcDNA组相比,miR-106a-5p+TLR4组的TLR4 mRNA及蛋白表达、48 h及72 h的细胞OD值、PCNA和Bcl-2蛋白表达明显增加(P<0.05);细胞凋亡率和Bax蛋白表达明显降低(P<0.05)。结论:miR-106a-5p通过下调TLR4抑制滑膜成纤维细胞MH7A的增殖,并诱导其细胞凋亡。

Objective:To investigate the effect of miR-106a-5p on the proliferation and apoptosis of rheumatoid arthritis(RA)synovial fibroblasts by regulating TLR4.Methods:The RA synovial fibroblasts MH7A were cul-tured in vitro,and were transfected with miR-NC,miR-106a-5p,si-NC,si-TLR4,anti-miR-NC,anti-miR-106a-5p,miR-106a-5p+pcDNA,or miR-106a-5p+TLR4.The expression of miR-106a-5p and TLR4 mRNA in the synovium of patients with RA was detected by qRT-PCR.Cell proliferation was assessed by MTT assay.Cell apoptosis was measured by flow cytometry.The expressions of TLR4,PCNA,Bax and Bcl-2 protein were deter-mined by Western blotting.The targeting relationship between miR-106a-5p and TLR4 was confirmed by dual lu-ciferase reporter assay.Results:The miR-106a-5p expression was down-regulated in RA synovial tissues,while the TLR4 mRNA and protein expressions were significantly up-regulated(P<0.05).Compared with miR-NC group,the miR-106a-5p expression,cell apoptosis rate and Bax protein expression were significantly increased in cells transfected with miR-106a-5p mimic,while the OD values at 48 h and 72 h,and the expressions of PCNA and Bcl-2 were markedly decreased(P<0.05).Compared with si-NC group,the TLR4 mRNA and protein ex-pression,OD values at 48 h and 72 h,and the expressions of PCNA and Bcl-2 were notably reduced,while the apoptosis rate and Bax protein level were obviously elevated in cells after interfering with TLR4 expression(P<0.05).miR-106a-5p mimic reduced the luciferase ac-tivity of TLR4-WT reporter but had no effect on the TLR4-MUT reporter in MH7A cells(P<0.05).Compared with anti-miR-NC group,the expression of miR-106a-5p was significantly decreased,and the TLR4 protein expression was significantly increased in anti-miR-106a-5p group(P<0.05).In cells co-transfected with miR-106a-5p mimic and pcDNA-TLR4 group,the ex-pression of TLR4 mRNA and protein,OD values at 48 h and 72 h as well as the PCNA and Bcl-2 protein expres-sions were increased,whereas the apoptosis rate and Bax protein expression were evidently decreased(P<0.05).Conclusion:miR-106a-5p inhibits the proliferation of synovial fibroblast MH7A cells and induces apoptosis by down-regulating TLR4.