miR-33a-5p靶基因分析验证及对人肺腺癌A549细胞增殖的影响  

Verification of miR-33a-5p Target Gene Analysis and Its Effect on Proliferation of Human Lung Adenocarcinoma A549 Cells

作　　者：吕治平 李靖[1] 何伟[1] 刘阳[1] 马云帆[1] 张升[1] 韩育宁[1] LYU Zhiping;LI Jing;HE Wei;LIU Yang;MA Yunfan;ZHANG Sheng;HAN Yuning(General Thoracic Surgery,the General Hospital of Ningxia Medical University,First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院普胸外科,宁夏医科大学第一临床医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第2期118-124,共7页Journal of Ningxia Medical University

基　　金：宁夏医科大学校级课题(xm2020164)。

摘　　要：目的研究miR-33a-5p过表达对人肺腺癌A549细胞增殖的影响及探索其可能的机制。方法在A549细胞中瞬时转染miR-33a-5p mimics,CCK-8和BrdU实验检测细胞增殖能力,转录组测序(RNA-seq)检测mRNA表达水平,targetscan(8.0)、miRDB(2020)、miRWalk(release_2022_01)和ENCORI(starbase,v2.0)联合筛选miR-33a-5p的潜在靶基因,并与RNA-seq的下调表达的基因取交集,RT-qPCR进行基因表达验证。结果RT-qPCR结果显示,miR-33a-5p在A549细胞中高表达(P<0.05);CCK-8实验和BrdU实验结果显示,细胞增殖能力受到抑制(P均<0.05);RNA-seq结果显示,差异基因共494个,其中上调266个,下调228个;GO功能富集主要在细胞外区组成、质膜的组成、受体复合物、细胞外泌体、细胞外基质结构组成、钙离子结合等,KEGG富集主要在补体和凝血途径、胰岛素抵抗、ABC转运途径、IL-17途径、NOD样受体途径和NF-κB信号通路等;靶基因预测分析和RT-qPCR表达验证显示,MTHFD2、OSBPL6、HADHB、HMGCLL1、GUCY1A2和DEPTOR是miR-33a-5p的潜在靶基因(P均<0.05)。结论miR-33a-5p可能通过调控MTHFD2、OSBPL6、HADHB、HMGCLL1、GUCY1A2和DEPTOR基因转录后表达水平来抑制A549细胞的增殖。Objective To investigate the effect of miR-33a-5p overexpression on the proliferation of human lung adenocarcinoma A549 cells and explore its possible mechanism.Methods A549 cells were transfected with miR-33a-5p mimics,CCK-8 and BrdU assay were applied to detect the cell proliferation.Transcriptome sequencing(RNA-seq)was used to detect transcriptome expression level.Targetscan(8.0),miRDB(2020),miRWalk(release_2022_01)and ENCORI(starbase,v2.0)were jointly used to screen potential target genes of miR-33a-5p,then intersected with differentially expressed genes of RNA-seq,and gene expression was verified by RT-qPCR.Results RT-qPCR showed that miR-33a-5p was overexpressed in A549 cells(P<0.05).CCK-8 assay and BrdU assay showed that cell proliferation was considerably inhibited(P all<0.05).The results of transcriptome sequencing showed 494 differential genes,266 of which were up-regulated and 228 down-regulated.GO functional enrichment was mainly in the extracellular region,integral component of plasma membrane and receptor complexes,extracellular exosomes,extracellular matrix structural constituent,and calcium ion binding,etc.KEGG enrichment occurred mainly in the complement and coagulation cascades,insulin resistance,ABC transporters,and IL-17 signaling pathway,NOD-like receptor signaling pathway,NF-κB signaling pathway,etc.Target gene prediction analysis and RT-qPCR expression verification showed that MTHFD2,OSBPL6,HADHB,HMGCLL1,GUCY1A2 and DEPTOR were significantly decreased(P all<0.05).Conclusion miR-33a-5p may inhibit the proliferation of A549 cells by regulating the post-transcriptional levels of MTHFD2,OSBPL6,HADHB,HMGCLL1,GUCY1A2,and DEPTOR genes.

关 键 词：miR-33a-5p 人肺腺癌A549细胞 转录组测序 

分 类 号：R734.2[医药卫生—肿瘤]