微小RNA-18a靶向Notch2抑制NIH-3T3细胞外基质相关基因的表达  

Inhibiting effect of miR-18a on expression of extracellular matrix-related genes of NIH-3T3 by targeting Notch2

作　　者：郝家祺 文静 刘春城 蔡禄 HAO Jiaqi;WEN Jing;LIU Chuncheng;CAI Lu(School of Life Science and Technology,Inner Mongolia University of Science and Technology,Baotou,Inner Monoglia 014010,China;Inner Mongolia Key Laboratory of Functional Genome Bioinformatics,Inner Mongolia University of Science and Technology,Baotou,Inner Monoglia 014010,China)

机构地区：[1]内蒙古科技大学生命科学与技术学院,内蒙古包头014010 [2]内蒙古科技大学内蒙古自治区功能基因组生物信息学重点实验室,内蒙古包头014010

出　　处：《环境与职业医学》2022年第7期786-791,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金(61671256,62071259);内蒙古科技大学创新基金项目(2019QDLB43,2019QDL-B44);内蒙古自治区高等学校科学研究项目(NJZY20096)。

摘　　要：[背景]矽肺主要病理特征是肺部纤维化。矽肺发生发展过程中多种miRNAs具有调控作用。[目的]利用成纤维细胞系,探究微小RNA-18a(miR-18a)对细胞外基质相关基因表达的影响,并对其作用机制进行验证。[方法]在成纤维细胞系NIH-3T3细胞中转染miR-18a模拟物以及神经源性基因座Notch同源蛋白2(Notch2)基因的小干扰RNA(siRNA)。通过应用实时定量逆转录聚合酶链反应(qRTPCR)技术检测Acta2、Col1a1及Notch2基因mRNA表达变化,通过蛋白质印迹技术检测α-平滑肌肌动蛋白(α-SMA)及Notch2蛋白的表达变化,利用双荧光素酶报告载体在人胚肾细胞HEK293T细胞中验证miR-18a调控Notch2基因的直接作用位点。[结果]qRT-PCR检测结果表明,在NIH-3T3细胞中,miR-18a模拟物过表达36 h抑制了Col1a1以及Acta2的m RNA表达(P<0.05),同时利用蛋白质印迹技术检测发现,miR-18a模拟物过表达48 h后α-SMA蛋白表达丰度降低。通过qRT-PCR未检测到过表达miR-18a 36 h对于Notch2基因表达的影响,通过蛋白质印迹技术检测发现过表达miR-18a模拟物36 h可以在蛋白水平上抑制Notch2的表达。双荧光素酶报告载体检测结果发现,在HEK293T细胞中,无论是过表达miR-18a模拟物或者抑制物24 h均证明了Notch2是miR-18a的直接靶基因。当Notch2被抑制36 h时,qRT-PCR检测发现Acta2和Col1a1基因表达下调(P<0.05);蛋白质印迹技术检测表明α-SMA在蛋白水平也受到抑制。[结论]本研究发现miR-18a可以通过直接作用于靶基因Notch2的3’UTR而抑制其表达,从而抑制成纤维细胞系NIH-3T3细胞外基质相关基因的表达。[Background]The main pathological feature of silicosis is pulmonary fibrosis.Multiple mi RNAs regulate the development of silicosis.[Objective]Using a fibroblast cell line,to explore the effect of mi R-18a on the expression of extracellular matrix-related genes,and verify the mechanism.[Methods]The fibroblast cell line NIH-3T3 cells were transfected with mi R-18a mimics or neurogenic locus notch homolog protein 2(Notch2)small interfering RNA(si RNA).The m RNA expression changes of Acta2,Col1a1,and Notch2 were detected by real-time quantitative reverse transcription PCR(q RT-PCR),α-smooth muscle actin(α-SMA)and Notch2 were also detected at the protein level by Western blotting.To verify whether mi R-18a could directly act on the complementary sequences of the Notch2 gene,human embryonic kidney HEK293T cells and the psi CHECKTM-2 vector were used.[Results]The results of q RT-PCR showed that in NIH-3T3 cells,the over-expression of mi R-18a mimics for 36 h inhibited the m RNA expression of Col1a1 and Acta2(P<0.05).The results of Western blotting showed that the protein expression abundance ofα-SMA was decreased at 48 h of mi R-18a mimics over-expression.The q RT-PCR results showed that the over-expression of mi R-18a for 36 h had no significant effect on Notch2 gene expression,but the Western blotting results showed that the over-expression of mi R-18a mimics inhibited the expression of Notch2 at the protein level.The results of the dual luciferase reporter vector assay showed that in HEK293T cells,both overexpressed mi R-18a mimics and inhibitors for 24 h demonstrated that Notch2 is a direct target gene of mi R-18a.When Notch2 was inhibited for 36 h,the q RT-PCR results showed that Acta2 and Col1a1 were down-regulated(P<0.05),and the results of Western blotting showed thatα-SMA protein was also inhibited.[Conclusion]The findings indicate that mi R-18a could inhibit the expression of extracellular matrix-related genes of NIH-3T3 cells by directly acting on the 3’UTR of target gene Notch2.

关 键 词：矽肺 微小RNA-18a 神经源性基因座Notch同源蛋白2 

分 类 号：R114[医药卫生—卫生毒理学]