慢病毒shRNA沉默Rac1表达抑制胃癌细胞上皮-间质转化的机制  

Mechanism of inhibiton of epithelial-mesenchymal transition in gastric cancer cells by silencing Rac1 expression by lentivirus-mediated short hairpin RNA

作　　者：张艺馨[1] 黄幼生[1,2] 解娜[1,2] 周晓明 王敏健[1] 吴文婷[1] 郑艺菲 ZHANG Yi-xin;HUANG You-sheng;XIE Na;ZHOU Xiao-ming;WANG Min-jian;WU Wen-ting;ZHENG Yi-fei(Department of Pathology,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,China;Department of Pathology,Hainan Medical University,Haikou 570102,China)

机构地区：[1]海南医学院第一附属医院病理科,海口570102 [2]海南医学院病理教研室,海口570102

出　　处：《临床与实验病理学杂志》2020年第10期1148-1154,共7页Chinese Journal of Clinical and Experimental Pathology

基　　金：海南省自然科学基金(818MS144)、海南医学院第一附属医院培育基金(HYFYPY201703)。

摘　　要：目的探讨Ras相关的C3肉毒素底物1(ras-related C3 botulinum toxin substrate 1,Rac1)促胃癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)的分子机制。方法应用TCGA数据及qPCR法分析Rac1基因在胃癌及其配对正常胃黏膜组织中的表达;慢病毒包装Rac1 shRNA沉默胃癌MGC-803细胞Rac1基因表达;CCK-8、细胞克隆实验检测Rac1基因表达沉默前、后胃癌细胞增殖、克隆能力的变化情况;qPCR、免疫组化、Western blot法检测Rac1基因沉默前、后EMT标志物、ERK1/2及p-ERK1/2表达变化。结果与配对正常胃黏膜组织相比,Rac1在胃癌组织中的表达增高(P<0.05),与淋巴结转移、浸润深度、TNM分期相关(P<0.05)。应用慢病毒沉默MGC-803细胞Rac1表达后,与阴性对照组相比,Rac1干扰组细胞增殖、克隆能力显著下调(P<0.05);胃癌细胞E-cadherin表达上调,N-cadherin、vimentin、Snail、MMP-9表达下调,ERK1/2磷酸化水平明显下降(P<0.05)。结论Rac1在胃癌组织中高表达,与胃癌浸润、转移、TNM分期有关;下调Rac1基因表达后能抑制胃癌细胞增殖及EMT,可能与阻断ERK1/2磷酸化有关。Purpose The present study aimed to investigate the effects of Rac1 knockdown on epithelial-mesenchymal transition(EMT),and to explore the possible molecular mechanism in gastric cancer.Methods The expression level of Rac1 gene was detected by qPCR method and TCGA(The Cancer Genome Atlas)data analysis in gastric cancer and its paired normal gastric mucosa tissues.Lentivirus-mediated short hairpin RNA(LV-shRNA)was used to knockdown Rac1 expression in gastric cancer cells MGC-803,and CCK-8,cell cloning experiments were used to detect the proliferation,cloning ability of gastric cancer cells respectively.Meanwhile,the expression level of EMT markers,ERK1/2 and p-ERK1/2 were detected by qPCR,immunohistochemistry and Western blot.Results The expression level of Rac1 in gastric cancer was higher than that in normal gastric mucosa(P<0.05),which was related to lymph node metastasis,invasion depth and TNM stage(P<0.05).After Rac1 gene was successful knocked out by infecting LV-shRNA in MGC-803 cells,compared with the negative control group,cell proliferation,cell cloning ability of the Rac1 interference group were significantly down-regulated(P<0.05).Furthermore,Western blot and IHC analysis indicated that vimentin,Snail,N-cadherin,MMP-9 were downregulated,whereas E-cadherin was upregulated,and ERK1/2 phosphorylation level was significantly decreased following Rac1 knockdown in gastric cancer MGC-803 cells.Conclusion Rac1 is highly expressed in gastric cancer tissues and is associated with invasion,metastasis and TNM stage of gastric cancer.Rac1 silencing can inhibit the proliferation and EMT of gastric cancer cells,which may be related to the blocking of ERK1/2 phosphorylation.

关 键 词：胃肿瘤 上皮-间质转化 RAC1 ERK1/2 转移 

分 类 号：R735.2[医药卫生—肿瘤]