LIMD2基因表达对食管癌细胞增殖、凋亡及ERK/MAPK信号通路的影响  

Effects of LIMD2 gene expression on the proliferation,apoptosis and ERK/MAPK signal pathway of esophageal cancer cells

作　　者：李险波 张志强[1] 韩小勇 甄志鹏 李焕芬 赵钦 LI Xian-bo;ZHANG Zhi-qiang;HAN Xiao-yong;ZHEN Zhi-peng;LI Huan-fen;ZHAO Qin(Department of Thoracic Surgery,Baoding First Central Hospital,Baoding Hebei 071000,China;Department of Thoracic Surgery,Hebei Provincial People’s Hospital,Shijiazhuang Hebei 050051,China)

机构地区：[1]保定市第一中心医院胸外科,河北保定071000 [2]河北省人民医院胸外科,河北石家庄050051

出　　处：《局解手术学杂志》2022年第8期669-675,共7页Journal of Regional Anatomy and Operative Surgery

基　　金：河北省医学科学研究课题计划(20190945)。

摘　　要：目的探讨LIM结构域2(LIMD2)对食管癌细胞增殖、凋亡的影响,及其对细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)通路的调控作用。方法取10例食管癌患者癌组织及癌旁组织标本,采用qRT-PCR检测LIMD2 mRNA表达,采用免疫荧光染色检测LIMD2蛋白表达,采用Western blot法检测ERK1/2、p-ERK1/2、p38MAPK、p-p38MAPK蛋白表达。取人食管癌细胞(KYSE30、EC9706)和人正常食管上皮细胞(HEEC),采用qRT-PCR及免疫荧光染色检测细胞中LIMD2 mRNA及蛋白表达,筛选出LIMD2 mRNA及蛋白表达最高的EC9706细胞进行后续试验。取EC9706细胞,分为对照组(常规培养)、Si-LIMD2组(转染LIMD2干扰序列的腺病毒载体)、Si-NC组(转染不含LIMD2干扰序列的空腺病毒载体)、PD98059组(ERK/MAPK通路阻断剂)、ISO组(ERK/MAPK通路激活剂)、Si-LIMD2+ISO组(在Si-LIMD2组基础上加入ISO溶液);采用qRT-PCR及免疫荧光染色检测细胞中LIMD2 mRNA及蛋白表达;采用CCK-8法检测细胞增殖活性;采用流式细胞术检测细胞周期及凋亡率;采用Western blot法检测细胞ERK1/2、p-ERK1/2、p38MAPK、p-p38MAPK、细胞周期相关蛋白cyclin D1和CDK2、caspase-3、整合素β1、黏着斑激酶(FAK)、整合素连接激酶(ILK)表达。结果LIMD2 mRNA及蛋白在食管癌组织及KYSE30细胞、EC9706细胞中的表达均较高,且在EC9706细胞中的表达高于KYSE30细胞,差异均有统计学意义(P<0.05);p-ERK1/2、p-p38MAPK蛋白在食管癌组织中表达较高(P<0.05)。与对照组相比,Si-LIMD2组和PD98059组FAK、ILK、整合素β1蛋白及p-ERK1/2、p-p38MAPK蛋白表达较低,增殖活性较低,G0/G1期比例较高,S期及G2/M期比例较低,凋亡率较高,cyclin D1、CDK2蛋白表达较低,caspase-3蛋白表达较高,差异均有统计学意义(P<0.05),ISO组FAK、ILK、整合素β1蛋白及p-ERK1/2、p-p38MAPK蛋白表达较高,增殖活性较高,G0/G1期比例较低,S期及G2/M期比例较高,凋亡率较低,cyclin D1、CDK2蛋白表达较高,caspase-3蛋白Objective To investigate the effects of LIM domain 2(LIMD2)on the proliferation and apoptosis of esophageal cancer cells,and the regulation on extracellular signal-regulated kinase(ERK)/mitogen-activated protein kinase(MAPK)pathway.Methods The samples of tumor tissues and adjacent tissues were taken from 10 patients with esophageal cancer.qRT-PCR was used to detect the expression of LIMD2 mRNA;immunofluorescence was used to detect the protein expression of LIMD2;Western blot was used to detect the protein expression of ERK1/2,p-ERK1/2,p38MAPK,and p-p38MAPK.Human esophageal cancer cell(KYSE30,EC9706)and normal human esophageal epithelial cell(HEEC)were taken.qRT-PCR and immunofluorescence were used to measure the expression of LIMD2 mRNA and protein in cells,and the EC9706 cell with the highest expression of LIMD2 mRNA and protein was selected for the follow-up experiments.EC9706 cells were taken and divided into the control group(routinely cultured),the Si-LIMD2 group(transfected with adenovirus vector containing LIMD2 interference sequence),the Si-NC group(transfected with empty adenovirus vector without LIMD2 interference sequence),the PD98059 group(ERK/MAPK pathway blocker),the ISO group(ERK/MAPK pathway activator),and the Si-LIMD2+ISO group(add ISO solution on basis of the si-LIMD2 group).qRT-PCR and immunofluorescence were used to measure the expression of LIMD2 mRNA and protein in cells;CCK-8 was used to measure cell proliferation activity;flow cytometry was used to measure cell cycle and apoptosis rate;Western blot was used to detect the expression of cell ERK1/2,p-ERK1/2,p38MAPK,p-p38MAPK,cell cycle related proteins cyclin D1 and CDK2,caspase-3,integrinβ1,focal adhesion kinase(FAK),integrin-linked kinase(ILK).Results LIMD2 mRNA and protein expression in esophageal cancer tissues,KYSE30 cells and EC9706 cells were higher,and the expression in EC9706 cells was higher than that in KYSE30 cells(P<0.05).The expression of p-ERK1/2 and p-p38MAPK protein in esophageal cancer tissues were higher(P<0.05).Compared

关 键 词：LIM结构域2 食管癌细胞 细胞外信号调节激酶 丝裂原活化蛋白激酶 增殖 凋亡 

分 类 号：R735.1[医药卫生—肿瘤]