REG1A通过调控Wnt/β-catenin信号通路对肺腺癌细胞增殖和迁移的促进作用  

Promotion effect of REG1A on proliferation and migration of lung adenocarcinoma cells by regulating Wnt/β-catenin signaling pathway

作　　者：李小燕 张维[1,3] 何杰 LI Xiaoyan;ZHANG Wei;HE Jie(School of Clinical Medical Sciences,Chengdu Medical College,Chengdu 610500,China;Department of Endocrinology,First Affiliated Hospital,Chengdu Medical College,Chengdu 610500,China;Department of Respiatory and Critical Care Medicine,First Affiliated Hospital,Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]成都医学院临床医学院,四川成都610500 [2]成都医学院第一附属医院内分泌科,四川成都610500 [3]成都医学院第一附属医院呼吸与危重症医学科,四川成都610500

出　　处：《吉林大学学报（医学版）》2022年第2期444-453,共10页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金青年科学基金项目(81600388)。

摘　　要：目的:探讨再生基因1A(REG1A)对肺腺癌细胞增殖和迁移的促进作用及其对Wnt/β-catenin信号通路的调控,并阐明可能的作用机制。方法:利用肿瘤基因组图谱(TCGA)数据库下载符合本研究要求的515例肺腺癌组织和59例癌旁正常组织的基因表达谱,根据REG1A表达水平的中位值,分为低和高表达REG1A组,采用R 3.6.1软件对2组基因进行差异基因分析,根据差异基因进行基因本体(GO)功能注释,采用GSEA 4.1.0软件筛查差异基因富集的京都基因与基因组百科全书(KEGG)信号通路。筛查出得分最高的信号通路[无翅型MMTV整合位点(Wnt)信号通路],采用实时荧光定量PCR(RT-qPCR)及Western blotting法检测人正常肺上皮细胞(BEAS-2B)和肺腺癌细胞A549、LC-2、HCC515和PC14中REG1A mRNA及蛋白表达水平。采用脂质体转染法将shREG1A和shNC质粒转染A549细胞,将REG1A敲减后的细胞,采用氯化锂(LiCl)处理,将细胞分为shNC组、shREG1A组、shREG1A+PBS组和shREG1A+LiCl组,采用细胞计数试剂盒(CCK-8)检测各组细胞增殖活性,采用Transwell实验检测各组细胞的迁移情况,RT-qPCR和Western blotting法检测各组细胞中无翅型MMTV整合位点家族成员3a(Wnt-3a)、β-连环素(β-catenin)和原癌基因(c-Myc)mRNA及蛋白表达水平。结果:生物信息学分析,低和高表达REG1A组共有78个差异基因,其中上调的基因50个,下调的基因28个,上述基因主要富集在脂质转运体活性、内切核糖核酸酶活性和胃肠道上皮结构稳态的维持等生物学过程和分子功能上,富集的通路有Wnt信号通路、Janus激酶-信号转导与转录激活因子(JAK-STAT)信号通路和癌症信号通路,其中Wnt信号通路富集得分最高。RT-qPCR法和Western blotting检测,肺腺癌A549、LC-2、HCC515和PC14细胞中REG1A mRNA及蛋白表达水平均明显高于BEAS-2B细胞(P<0.05)。脂质体转染法敲减REG1A后,与shNC组比较,shREG1A组A549细胞中REG1A mRNA和蛋白表达水平明显降低(P<0.Objective:To investigate the promotion effect of regenerating gene 1A(REG1A)on the proliferation and migration of lung adenocarcinoma cells and its regulation effect on Wnt/β-catenin signaling pathway,and to clarify its possible mechanism.Methods:The gene expresson profiles of 515 patients with lung adenocarcinoma and 59 paracancerous normal lung tissue met the requirement of this study were obtained from The Cancer Genome Atlas(TCGA)database.According to the median value of the REG1A distribution level,they were divided into high and low expression groups.R 3.6.1 software was employed to analyze the differentially expressed genes between two groups.The Gene Ontology(GO)function annotation was carried out for the differential genes.Meanwhile,GSEA4.1.0 was used for Kyoto Encyclopedia of Genes and Genomes(KEEF)enrichment analysis of the differential genes.Wnt signaling pathway,the signaling pathway that scored the best,was screened,and the mRNA and the protein expression levels of REG1A in the human normal lung epithelial cells(BEAS-2B)and lung adenocarcinoma cells(A549,LC-2,HCC515 and PC14)were tested by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.The liposome transfection method was used to transfect shREG1A and shNC into the A549 cells,and the A549 cells with REG1A expression knockdown were treated by lithium chloride.The A549 cells were divided into shNC group,shREG1A group,shREG1A+PBS group,and shREG1A+LiCl group.The proliferation activities of the cells in various groups were detected by CCK-8 method.The migration of cells in various groups was detected by Transwell assay.The mRNA and proein expression levels of Wnt-3a,β-catenin,and c-Myc in the cells in various groups were detected with RT-qPCR and Western blotting methods.Results:The 78 differential genes in low and high expression REG1A groups were screened out,including 50 up-regulated genes and 28 down-regulated genes;these genes were mainly enriched in proton antiporter activity,endoribonuclease activity,maintenance of

关 键 词：肺腺癌 再生基因1A 细胞增殖 细胞迁移 生物信息学 

分 类 号：R734.2[医药卫生—肿瘤]