加权基因共表达网络分析微RNA-92b-3p在食管鳞状细胞癌中的作用及机制  

Role and mechanism of microRNA-92b-3p in esophageal squamous cell carcinoma analyzed by weighted gene co-expression network analysis

作　　者：王万鹏 傅承宏 张启迪 王成师[1] 何中祥 顾云[1] 张艳艳 邓卫军[3] 濮娟 Wang Wanpeng;Fu Chenghong;Zhang Qidi;Wang Chengshi;He Zhongxiang;Gu Yun;Zhang Yanyan;Deng Weijun;Pu Juan(Department of Radiotherapy, Lianshui People′s Hospital, Huai′an City 223400, Jiangsu Province, China;Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiaotong University College of Medicine, Shanghai 200080, China;Department of Thoracic Surgery, Lianshui People′s Hospital, Huai′an City 223400, Jiangsu Province, China)

机构地区：[1]涟水县人民医院放疗科,江苏省淮安市223400 [2]上海交通大学医学院附属上海市第一人民医院消化科,200080 [3]涟水县人民医院胸外科,江苏省淮安市223400

出　　处：《中华消化杂志》2019年第6期390-396,共7页Chinese Journal of Digestion

基　　金：国家自然科学基金青年项目(81600479、81600549);江苏省卫生健康委科研项目(Z2018026);淮安市自然科学研究计划(HAB201737).

摘　　要：目的通过加权基因共表达网络分析(WGCNA)对食管鳞状细胞癌(ESCC)发展相关基因进行筛选,并通过实验验证。方法以基因芯片平台(GPL)570、GPL571、GPL96/97或GPL14613为基础,从基因表达数据库(GEO)中下载ESCC基因表达谱数据集;将所得差异基因结合癌症基因组图谱(TCGA)的81例ESCC患者的基因表达谱数据和临床信息,通过WGCNA方法构建由mRNA和miRNA组成的共表达模块。实时荧光定量PCR检测ESCC癌组织和癌旁组织中miRNA表达,免疫组织化学法检测Kruppel样因子4(KLF4)和细胞桥粒胶蛋白2(DSC2)的表达;转染ESCC细胞系ECA-109 miRNA模拟物后,流式细胞术检测细胞周期,Transwell小室和划痕实验检测细胞侵袭和迁移能力,激光共聚焦显微镜和蛋白质印迹法检测KLF4和DSC2的表达,双荧光素酶报告基因系统验证靶基因。统计学分析采用t检验、秩和检验、卡方检验和Pearson相关分析。结果共筛选出4 023个差异蛋白编码基因(DEG)和328个差异表达微RNA(DEM);通过WGCNA共构建出11个基因模块。其中brown模块与肿瘤分级和T分期均呈负相关(r=-0.340、-0.268,P=0.002、0.016),has-mir-92b及其潜在靶基因KLF4和DSC2同时包含于brown模块。实时荧光定量PCR显示,ESCC组织中miRNA-92b-3p(has-miR-92b成熟体)表达水平高于癌旁组织[3.052(1.652, 5.371)比0.985(0.558, 2.032)],差异有统计学意义(Z=-4.021,P<0.01)。免疫组织化学显示KLF4和DSC2在ESCC组织中的阳性率分别为43.3%(13/30)和20.0%(6/30),分别低于对应癌旁组织的70.0%(21/30)和63.3%(19/30),差异均有统计学意义(χ^2=4.344、11.589,P均<0.05)。转染miRNA-92b-3p模拟物后,ECA-109细胞G0/G1期缩短[(63.71±2.83)%比(54.62±4.00)%],S期和G2/M期均延长[分别为(31.81±2.88)%比(41.20±2.87)%,(3.87±1.75)%比(8.10±1.71)%],差异均有统计学意义(t=3.215、4.000、2.998,P=0.032、0.016、0.040)。细胞侵袭和迁移能力显著增强[分别为79.67±27.54比280.33±46.18,(69.72±3.91)%比(84.90±5Objective To screen the critical genes related to the development of esophageal squamous cell carcinoma (ESCC) by weighted gene co-expression network analysis (WGCNA) and to verify by experiments. Methods Gene expression data of ESCC were downloaded from gene expression omnibus (GEO) database based on gene chip platform (GPL) 570, GPL571, GPL96/97 or GPL14613 platform, respectively. Meanwhile, the obtained differentially expressed genes together with gene expression data of 81 ESCC patients from the cancer genome atlas (TCGA) and clinical data were analyzed by WGCNA to set up co-expression networks including mRNA and microRNA (miRNA). The expression of miRNA in ESCC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction (RT-PCR). And the expression of target protein Kruppel like factor 4 (KLF4) and desmocollin 2 (DSC2) were detected by immunohistochemistry. After ESCC cell line ECA-109 cells were transfected with miRNA-92b-3p mimic, cell cycle was tested by flow cytometry, the cell invasion and migration ability was measured by Transwell chamber assay and scratch-wound assay. The expression of KLF4 and DSC2 was observed by confocal laser scanning microscopy and Western blotting.The target genes were verified by luciferase assay. T-test, rank sum test, chi-square test and Pearson correlation analysis were performed for statistical analysis. Results A total of 4 023 differential expression gene (DEG) and 328 differential expression miRNA (DEM) were screened and 11 gene modules were set up by WGCNA. Among them, the brown modules were negatively associated with tumor grade and T stage (r=-0.340 and -0.268, P=0.002 and 0.016). Meanwhile, has-miR-92b and the potential target genes KLF4 and DSC2 were all in the brown module. Furthermore, the results of RT-PCR showed the expression of miRNA-92b-3p in ESCC tissues was higher than that in paracancerous tissues (3.052(1.652, 5.371) vs. 0.985(0.558, 2.032)), and the difference was statistically significant (Z=-4.021, P<0.01). The r

关 键 词：计算生物学 加权基因共表达网络分析 食管鳞状细胞癌 微RNA-92b Kruppel样因子4 桥粒胶蛋白2 

分 类 号：R735.1[医药卫生—肿瘤]