miR-155对人胚肺成纤维细胞HELF增殖、凋亡和胶原蛋白合成的影响

Effects of miR-155 on proliferation, apoptosis and collagen synthesis of human embryonic lung fibroblasts cell HELF

目的:探讨miR-155对人胚肺成纤维细胞HELF增殖、凋亡和胶原蛋白合成的影响。方法:通过转染miR-155抑制剂构建miR-155低表达的HELF细胞株,采用qRT-PCR检测转染效果,CCK-8法和流式细胞仪检测miR-155低表达对HELF细胞增殖、凋亡的影响,Western blot检测miR-155低表达对Col-Ⅰ和Col-Ⅲ胶原蛋白表达的影响。采用TargetScan软件预测并通过双荧光素酶报告基因实验检测miR-155和Smad5的靶向关系,qRT-PCR和Western blot检测miR-155和Smad5的调控关系。将si-Smad5和miR-155抑制剂共同转染HELF细胞后,观察Smad5沉默是否影响miR-155对HELF细胞增殖、凋亡和胶原蛋白合成的作用。结果:转染miR-155抑制剂成功下调了HELF细胞中miR-155的表达,miR-155低表达能够明显抑制HELF细胞增殖和Col-Ⅰ、Col-Ⅲ胶原蛋白的合成,并促进HELF细胞凋亡(P<0.05)。miR-155可与Smad53′UTR区域结合并负向调控Smad5表达(P<0.05)。Smad5沉默可逆转miR-155低表达对HELF细胞增殖、凋亡和胶原蛋白合成的调控作用(P<0.05)。结论:miR-155低表达可通过靶向调控Smad5抑制HELF细胞增殖和胶原蛋白合成,并促进HELF细胞凋亡。

Aim:To investigate the effects of miR-155 on proliferation,apoptosis and collagen synthesis of human embryonic lung fibroblasts cell HELF.Methods:HELF cell lines with low expression of miR-155 were constructed by transfecting miR-155 inhibitor.The transfection effect was examed by qRT-PCR,the effects of low expression of miR-155 on proliferation and apoptosis of HELF cells were measured by CCK-8 and flow cytometry,and the effect of low expression of miR-155 on expressions of collagen Col-Ⅰand Col-Ⅲwas detected by Western blot.The targeting relationship between miR-155 and Smad5 was predicted by TargetScan software,and was detected by double luciferase reporter gene assay.The regulatory relationship between miR-155 and Smad5 were tested by qRT-PCR and Western blot.si-Smad5 and miR-155 inhibitor were co-transfected into HELF cells so as to observe whether Smad5 silencing affected miR-155 on proliferation,apoptosis and collagen synthesis of HELF cells.Results:The expression of miR-155 in HELF cells was successfully down-regulated by transfection of miR-155 inhibitor,the proliferation of HELF cells and the synthesis of Col-Ⅰand Col-Ⅲwere inhibited,and the apoptosis of HELF cells were promoted by the low expression of miR-155(P<0.05).Smad53′UTR region could be combined with miR-155,and its expression could be negatively regulated by miR-155(P<0.05).The effects of low expression of miR-155 on proliferation,apoptosis and collagen synthesis of HELF cells could be reversed by Smad5 silencing(P<0.05).Conclusion:The low expression of miR-155 can inhibit the proliferation and collagen synthesis of HELF cells and promote the apoptosis of HELF cells through targeted regulation of Smad5.