LncRNA GAS5通过吸附miR-21促进ADAMTS-1介导的肺成纤维细胞Ⅰ、Ⅲ型胶原降解

LncRNA GAS5 promotes ADAMTS-1-mediated degradation of collagen types Ⅰ and Ⅲ by adsorbing miR-21 in pulmonary fibroblasts

目的观察生长阻滞特异性转录本5(growth arrest-specific transcript 5,GAS5)对肺成纤维细胞Ⅰ型胶原(ColⅠ)和Ⅲ型胶原(ColⅢ)表达的影响,并探讨其机制。方法培养人胚肺成纤维细胞MRC-5,用GAS5慢病毒过表达载体(LV-GAS5)处理,荧光实时定量PCR(qRT-PCR)检测GAS5、miR-21水平,qRT-PCR和Western blot测定I型血小板结合蛋白基序的解聚蛋白样金属蛋白酶-1(ADAMTS-1)、ColⅠ、ColⅢ表达。生物信息学、双荧光素酶报告基因分析GAS5、ADAMTS-1 3′-非翻译区与miR-21的靶向结合情况。以miR-21模拟物(mimic)或ADAMTS-1 siRNA转染MRC-5细胞48 h,再用LV-GAS5处理细胞相同时间,qRT-PCR和Western blot检测ADAMTS-1、ColⅠ、ColⅢ表达。结果过表达GAS5降低miR-21水平,抑制ColⅠ、ColⅢ蛋白表达,增加ADAMTS-1 mR NA与蛋白水平(P <0.01)。GAS5、miR-21、ADAMTS-1之间存在竞争性内源RNA (ceR NA)作用模式。miR-21 mimic或ADAMTS-1 siR NA预处理几乎完全逆转过表达GAS5对ColⅠ、ColⅢ降解的促进作用(P <0.01)。结论 GAS5作为ceR NA竞争性结合miR-21,上调ADAMTS-1表达,从而促进MRC-5细胞ColⅠ、ColⅢ降解。

Aim To observe the effects of growth arrest-specific transcript 5(GAS5)on collagen type I(ColⅠ)and collagen typeⅢ(ColⅢ)expression in pulmonary fibroblasts and explore the underlying mechanisms.Methods Human pulmonary fibroblasts MRC-5 were cultured and then transducted with lentiviral vector expressing GAS5(LV-GAS5).The expression levels of GAS5 and miR-21 were detected using quantitative real-time PCR(qRT-PCR).Both qRT-PCR and Western blot were used to measure a disintegrin-like and metalloproteinase with thrombospondin type 1 motif(ADAMTS-1),ColⅠand ColⅢexpression.Bioinformatics and dual luciferase reporter gene analyzed the binding of GAS5 and ADAMTS-13′-untranslated region to miR-21.In addition,MRC-5 cells were transfected with miR-21 mimic or ADAMTS-1 siRNA for 48 h,followed by treatment with LV-GAS5 for the same time.The expression of ADAMTS-1,ColⅠand ColⅢwas then detected by qRT-PCR and Western blot.Results Overexpression of GAS5 significantly decreased miR-21 levels,inhibited ColⅠand ColⅢprotein expression,and elevated the mRNA and protein levels of ADAMTS-1(P<0.01).There was an action mode of competitive endogenous RNA(ceRNA)among GAS5,miR-21,and ADAMTS-1.Furthermore,pretreatment with miR-21 mimic or ADAMTS-1 siRNA almost completely reversed the promoting effect of GAS5 overexpression on ColⅠand ColⅢdegradation in MRC-5 cells(P<0.01).Conclusions GAS5 as a ceRNA competitively binds to miR-21,leading to up-regulation of ADAMTS-1 expression and subsequent degradation of ColⅠand ColⅢin MRC-5 cells.