长链非编码lncRNA DBH⁃AS1对瘢痕疙瘩成纤维细胞凋亡的影响及作用机制

Impact of long non⁃coding lncRNA DBH⁃AS1 on apoptosis of keloid fibroblasts and its mechanism of action

目的探讨长链非编码lncRNA DBH⁃AS1(DBH⁃AS1)对瘢痕疙瘩成纤维细胞凋亡的影响及机制。方法采用实时荧光定量逆转录聚合酶链反应(qRT⁃PCR)检测瘢痕疙瘩和正常皮肤组织中DBH⁃AS1、MicroRNA⁃138(miR⁃138)和Hypoxia⁃inducible factor⁃1α(HIF⁃1α)的mRNA表达水平。采用双荧光素酶报告分析三者的结合情况。采用流式细胞技术、qRT⁃PCR和免疫印迹法(Western blot)检测敲低或过表达DBH⁃AS1对瘢痕疙瘩成纤维细胞凋亡的影响,并分析miR⁃138和HIF⁃1α的表达变化。结果DBH⁃AS1和HIF⁃1α瘢痕疙瘩组织和细胞中显著上调,而miR⁃138明显下调(P<0.05)。双荧光素酶结果显示DBH⁃AS1能够靶向结合miR⁃138,miR⁃138能够靶向结合HIF⁃1α。低表达DBH⁃AS1或过表达miR⁃138明显促进瘢痕疙瘩成纤维细胞凋亡。敲低miR⁃138能逆转沉默DBH⁃AS1对瘢痕疙瘩成纤维细胞凋亡的影响,敲低HIF⁃1α显著抑制沉默miR⁃138对细胞凋亡的作用(P<0.05)。结论低表达DBH⁃AS1能够通过调控miR⁃138/HIF⁃1α通路促进瘢痕疙瘩成纤维细胞凋亡,提示DBH⁃AS1可能成为瘢痕疙瘩治疗的一个潜在靶点。

Objective To explore the effect of long non⁃coding lncRNA DBH⁃AS1(DBH⁃AS1)on apoptosis of keloid fibroblasts and its mechanism of action.Methods Real⁃time fluorescent quantitative reverse transcription polymerase chain reaction(qRT⁃PCR)was used to detect mRNA expression levels of DBH⁃AS1,microRNA⁃138(miR⁃138)and Hypoxia⁃inducible factor⁃1α(HIF⁃1α)in keloids and normal skin tissues.Dual Luciferase Report was applied to analyze the combination of the three proteins.Flow cytometry,qRT⁃PCR and Western blot were utilized to detect the effect of knockdown or overexpression of DBH⁃AS1 on apoptosis of keloid fibroblasts,the expression changes of miR⁃138 and HIF⁃1αwere also analyzed.Results DBH⁃AS1 and HIF⁃1αwere significantly up⁃regulated in keloid tissues and cells while miR⁃138 was significantly down⁃regulated(P<0.05).The Dual Lucif⁃erase Report verified that DBH⁃AS1 could have targeted binding with miR⁃138,and miR⁃138 could have targeted binding with HIF⁃1αin keloid fibroblast.Low expression of DBH⁃AS1 or overexpression of miR⁃138 significantly promoted apoptosis of keloid fibroblasts.Knockdown of miR⁃138 could reverse the effect of silencing DBH⁃AS1 on apoptosis of keloid fibroblasts,and knockdown of HIF⁃1αsignificantly inhibited the effect of silencing miR⁃138 on apoptosis(P<0.05).Conclusions Silencing DBH⁃AS1 promoted apoptosis of keloid fibroblasts by regulating the miR⁃138/HIF⁃1αpathway,suggesting that DBH⁃AS1 may become a potential target for the treatment of keloids.