下调lncRNA-H19表达促进人瘢痕疙瘩成纤维细胞凋亡和自噬

Down-regulation of lncRNA-H19 expression promotes apoptosis and autophagy of human keloid fibroblasts

目的探讨下调长链非编码RNA-H19(lncRNA-H19)表达对人瘢痕疙瘩成纤维细胞系凋亡和自噬的影响,并初步探讨其作用机制。方法取人类瘢痕成纤维细胞系(KFs)及健康人皮肤成纤维细胞系(HDFs),将KF细胞感染lncRNA-H19F低表达腺病毒(si-lncRNA-H19)及不含si-lncRNA-H19的空病毒载体(si-eGFP)。分为HDFs组、KF组、si-lncRNA-H19+KFs组、si-eGFP+KFs组;KFs细胞共感染si-lncRNA与mTOR过表达腺病毒(Ad-mTOR)及空载体(Ad-eGFP-m),分为KFs组、si-lncRNA-H19+Ad-mTOR组、si-eGFP+Ad-mTOR组、si-lncRNA-H19+Ad-eGFP组、si-eGFP+Ad-eGFP组。RT-qPCR检测lncRNA-H19表达;流式细胞测量术检测细胞凋亡率;透射电镜及吖啶橙(Ao)荧光染色观察自噬形成情况;免疫组化法检测自噬通路-雷帕霉素靶蛋白(mTOR)阳性表达;Western blot检测凋亡及自噬相关蛋白表达。结果与HDFs组比较,KFs组细胞lncRNA-H19表达及mTOR活性升高,自噬泡数目、凋亡与自噬相关蛋白表达降低(P<0.05);下调KFs细胞中的lncRNA-H19后,KFs细胞mTOR活性降低,凋亡及自噬活性升高(P<0.05)。下调lncRNA-H19的同时,上调mTOR,KFs细胞的自噬及凋亡活性较单独下调lncRNA-H19时升高(P<0.05)。结论下调lncRNA-H19表达,可能通过抑制mTOR途径活化,促进KFs细胞自噬及凋亡。

Objective To investigate the effects of down-regulation of long non-coding RNA-H19(lncRNA-H19)expression on apoptosis and autophagy of human keloid fibroblast cell line and to explore potential mechanism.Methods Human scar fibroblasts cell line(KFs)and normal human dermal fibroblasts cell line(HDFs)were collected and KFs cells were infected with lncRNA-H19F low-expressing adenovirus(si-lncRNA-H19)and empty virus vector without si-lncRNA-H19(si-eGFP).They were divided into HDFs group,KFs group,si-lncRNA-H19+KFs group and si-eGFP+KFs group.KFs cells were co-transfected with si-lncRNA,mTOR over-expressing adenovirus(Ad-mTOR)and empty vector(Ad-eGFP-m),then divided into KFs group,si-lncRNA-H19+Ad-mTOR group,si-eGFP+Ad-mTOR group,si-lncRNA-H19+Ad-eGFP group and si-eGFP+Ad-eGFP group.RT-qPCR was used to detect the expression of lncRNA-H19;flow cytometry was used to detect the cell apoptosis;autophagy was examined by EM microscopy with fluorescence(acridine orange,AO)staining;immuno-histochemical method was used to detect the positive expression of autophagy pathway-mammalian target of rapamycin(mTOR);Western blot was used to detect the expression of apoptosis and autophagy-related proteins.Results Compared with the HDFs group,the expression of lncRNA-H19 and mTOR activity in the KFs group increased and the number of autophagic vesicles and the expression of apoptosis and autophagy-related proteins decreased(P<0.05);after down-regulating lncRNA-H19 in KFs cells,the activity of mTOR in KFs cells decreased and apoptosis/autophagy increased(P<0.05).Up-regulating mTOR while down-regulating lncRNA-H19 lead to more autophagy and apoptotic in KFs cells as compared to that resulted from down-regulation of lncRNA-H19 alone(P<0.05).Conclusions Down-regulation of lncRNA-H19 expression may promote the autophagy and apoptosis of KFs cells through inhibiting activation of mTOR pathway.