摘要
目的探讨微小RNA(miRNA,miR)-484靶向肝细胞核因子1A(HNF1A)调控食管癌恶性细胞行为的分子机制。方法选取河南省人民医院2017年6月至2019年1月手术切除的101例食管癌组织和癌旁组织作为研究对象,采用荧光定量聚合酶链式反应(Real-time PCR)分析miR-484表达水平;采用慢病毒介导miRNA对照和miR-484在食管癌细胞系Eca109建立miRNA对照组和miR-484组过表达细胞系。采用细胞计数试剂盒(CCK-8)分析两组细胞增殖能力;采用Transwell分析两组细胞迁移能力;采用流式细胞术分析两组细胞凋亡;采用生物信息学和双荧光素酶报告基因分析miR-484靶基因,采用蛋白质免疫印迹法(Western blot)分析靶基因及迁移和凋亡相关蛋白表达。组间比较采用t检验。结果癌旁组织miR-484表达水平(1.09±0.21)高于食管癌组织(0.31±0.17),差异有统计学意义(t=3.117,P<0.05)。miRNA对照组细胞miR-484表达水平(1.21±0.17)低于食管癌组织(3.96±0.35),差异有统计学意义(t=3.117,P<0.05)。miRNA对照组24 h和48 h吸光度(A)值(1.14±0.14、1.93±0.22)高于miR-484组细胞24 h和48 h A值(0.81±0.13、1.48±0.27),差异有统计学意义(t=2.910、2.791,P<0.05),miRNA对照组细胞侵袭数量[(74.03±5.90)个]高于miR-484组细胞侵袭数量[(37.84±3.99)个],差异有统计学意义(t=4.805,P<0.05)。miRNA对照组细胞凋亡比例[(5.09±1.73)%]低于miR-484组细胞[(20.43±4.08)%],差异有统计学意义(t=3.719,P<0.05)。HNF1A是miR-484的靶基因。miRNA对照组细胞HNF1A和Caspase-3蛋白相对表达水平(1.89±0.25、0.63±0.12)低于miR-484组细胞(5.11±0.72、1.06±0.15),差异有统计学意义(t=6.109、2.091,P<0.05)。miRNA对照组细胞FAK蛋白相对表达水平(1.97±0.28)高于miR-484组细胞(1.48±0.21),差异有统计学意义(t=2.018,P<0.05)。结论miR-484在食管癌组织中呈低表达,通过影响靶蛋白HNF1A表达水平,参与食管癌细胞增殖、迁移和凋亡等生物学行为。
Objective To explore the molecular mechanism of microRNA(miRNA,miR)-484 targeting hepatocyte nuclear factor 1-alpha(HNF1A)in regulating the behavior of esophageal cancer cells.Methods Esophageal cancer tissues and adjacent tissues from June 2017 to January 2019(a total of 101 cases)in the Henan people′s hospital were selected as the research objects.The expression level of miR-484 was analyzed by polymerase chain reaction(PCR).The overexpression cell lines of miRNA control group and miR-484 group were established by lentivirus mediated miRNA control and miR-484 in esophageal cancer cell line Eca109.The proliferation ability of the two groups was analyzed by cell counting kit-8(CCK-8)assay.The migration ability of the two groups was analyzed by transwell.The apoptosis rate in the two groups was analyzed by flow cytometry.miR-484 target genes were analyzed by bioinformatics and dual luciferase reporter genes.The expression of target genes and migration and apoptosis related proteins was analyzed by Western blotting.SPSS 17.0 statistical software was used to analyze the data.The data in accordance with normal distribution were expressed as mean±standard deviation.Results The expression level of miR-484 in adjacent tissues(1.09±0.21)was significantly higher than that in esophageal carcinoma tissues(0.31±0.17,t=3.117,P<0.05).The expression level of miR-484 in miRNA control group(1.21±0.17)was significantly lower than that in esophageal cancer(3.96±0.35)(t=3.117,P<0.05).The 24-h and 48-h absorbance(A)in miRNA control group(1.14±0.14,1.93±0.22)was significantly higher than that in miR-484 group(0.81±0.13,1.48±0.27,t=2.910,P<0.05).The number of invasive cells in miRNA control group(74.03±5.90)was significantly greater than that in miR-484 group(37.84±3.99,t=4.805,P<0.05).The apoptosis rate in miRNA control group[(5.09±1.73)%]was significantly higher than that in miR-484 group[(20.43±4.08)%,t=3.719,P<0.05].HNF1A is the target gene of miR-484.The relative expression levels of HNF1A and Caspase-3 protein in m
作者
陈晓汤
金星
魏立
Chen Xiao;Tang Jinxing;Wei Li(Department of Thoracic Surgery,Henan Provincial People's Hospital(The People's Hospital of Zhengzhou University),Zhengzhou 450003,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第4期680-683,共4页
Chinese Journal of Experimental Surgery
关键词
微小RNA
食管癌
增殖
迁移
凋亡
MicroRNA
Esophageal cancer
Proliferation
Migration
Apoptosis
