LncRNA MRAK052509对矽尘诱导RLE-6TN中TGF-β信号通路与纤维化影响  

Effects of lncRNA MRAK052509 on TGF-βsignaling pathway and fibrosis in silica dust-induced RLE-6TN cells

作　　者：刘煊 高芳瑜 陈溪渊 田雪艳 焦子铭 侯朋邑 王发选 LIU Xuan;GAO Fang-yu;CHEN Xi-yuan;TIAN Xue-yan;JIAO Zi-ming;HOU Peng-yi;WANG Fa-xuan(School of Public Health and Management,Ningxia Medical University,Ningxia Key Laboratory of Environmental Factors and Chronic Disease Control,Yinchuan,Ningxia 750004,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院,宁夏环境因素与慢性病控制重点实验室,宁夏银川750004

出　　处：《中国职业医学》2022年第1期8-14,共7页China Occupational Medicine

基　　金：国家自然科学基金(82060584,81660534);宁夏自然科学基金(2020AAC02019);宁夏高等教育科学研究项目(NGY2020035)。

摘　　要：目的探讨长链非编码RNA(lncRNA)MRAK052509对矽尘诱导的大鼠肺泡Ⅱ型上皮细胞株RLE-6TN中转化生长因子-β(TGF-β)/SMAD信号通路及下游相应纤维化因子表达的影响。方法(1)建立大鼠肺泡巨噬细胞株NR8383/RLE-6TN细胞共培养体系,随机分为对照组和模型组,模型组予剂量为400 mg/m^(2)的游离二氧化硅刺激,采用实时荧光定量聚合酶链反应(qPCR)法检测2组RLE-6TN中lncRNA MRAK052509相对表达水平。(2)将RLE-6TN随机分为对照组、阴性对照组和敲减组,后2组分别以阴性对照病毒和lncRNA MRAK052509敲减病毒转染,以qPCR法检测转染效率。(3)将NR8383/RLE-6TN细胞共培养体系随机分为4组,对照组和矽肺模型组下室均种入RLE-6TN细胞,阴性对照组和敲减组下室分别种入稳定转染阴性对照病毒和lncRNA MRAK052509敲减病毒的RLE-6TN细胞。除对照组外,其余3组向Transwell上室加入剂量为400 mg/m^(2)的游离二氧化硅刺激24 h后,收集RLE-6TN。采用qPCR法检测RLE-6TN中TGF-βⅠ型受体(TGF-βRⅠ)、Smad3和纤维黏连蛋白(FN)的mRNA相对表达水平,以蛋白免疫印迹法检测RLE-6TN中TGF-βRⅠ、SMAD3、磷酸化SMAD3(p-SMAD3)、FN、Ⅰ型胶原蛋白(COLⅠ)和Ⅲ型胶原(COLⅢ)蛋白相对表达水平。结果(1)模型组RLE-6TN细胞的lncRNA MRAK052509 mRNA表达水平较对照组升高(P<0.01)。(2)敲减组RLE-6TN中lncRNA MRAK052509 mRNA相对表达水平分别低于空白对照组和阴性对照组(P值均<0.05)。(3)与对照组比较,模型组RLE-6TN中TGF-βRⅠ、Smad3、FN的mRNA相对表达水平和TGF-βRⅠ、SMAD3、p-SMAD3、FN、COLⅠ、COLⅢ蛋白相对表达水平均升高(P值均<0.05);与矽肺模型组和阴性对照组比较,敲减组RLE-6TN中上述指标相对表达水平均下降(P值均<0.05)。结论敲减LncRNA MRAK052509可通过抑制TGF-βRⅠ的表达,阻断TGF-β/SMAD信号通路,延缓SiO2刺激所致RLE-6TN间质转化,下调FN、COLⅠ和COLⅢ等胶原纤维的表达,影响肺纤维化的进展�Objective To investigate the effect of long non-coding RNA(lncRNA)MRAK052509 on the expression of transforming growth factor-β(TGF-β)/SMAD signaling pathway and its downstream fibrogenic factors in silica-induced rat alveolar typeⅡepithelial RLE-6 TN cells.Methods i)The co-culture system consisting of rat alveolar macrophage NR8383 and RLE-6 TN cells was established and randomly divided into control group and silicosis model group.The silicosis model group was stimulated with 400 mg/m^(2) free silica.The relative expression of lncRNA MRAK052509 in RLE-6 TN cells was detected by real-time quantitative polymerase chain reaction(qPCR).ii)RLE-6 TN cells were randomly divided into control group,negative control group and lncRNA MRAK052509 knockdown group.The latter two groups were transfected with negative control virus and lncRNA MRAK052509 knockdown virus.The transfection efficiency was detected by qPCR.iii)NR8383/RLE-6 TN cells co-culture system was randomly divided into four groups.RLE-6 TN cells were cultured in the lower compartment of the control group and silica dust group,and RLE-6 TN cells stably transfected with short hairpin RNA-unintentional control and short hairpin RNA-lncRNA MRAK052509 in the lower compartment of the negative control group and knockdown group,respectively.Except for the control group,the cells in the other three groups were stimulated with 400 mg/m^(2) free silica.The RLE-6 TN cells in the lower compartment were collected after 24 hours.The qPCR was used to detect the relative expression of TGF-βtypeⅠreceptor(TGF-βRⅠ),Smad3 and fibronectin(FN)mRNA in the RLE-6 TN cells.The protein relative expression of TGF-βRⅠ,SMAD3,phospho-SMAD3(p-SMAD3),FN,collagen typeⅠ(COLⅠ)and collagen typeⅢ(COLⅢ)in RLE-6 TN cells was measured by Western blotting.Results i)The expression of lncRNA MRAK052509 mRNA in RLE-6 TN cells of silicosis model group was higher than that in control group(P<0.01).ii)The relative expression of lncRNA MRAK052509 mRNA in RLE-6 TN cells in knockdown group was

关 键 词：肺纤维化 lnc RNA 转化生长因子-βⅠ型受体 SMADS 纤维黏连蛋白 胶原蛋白 信号通路 

分 类 号：R135.2[医药卫生—劳动卫生] R114[医药卫生—公共卫生与预防医学]