SMC4在贲门腺癌中的作用及机制  

Role and mechanism of SMC4 in cardia adenocarcinoma

作　　者：朱梦琪 张懿刚 冯晓佳 张凌梅 贾建光[1] ZHU Mengqi;ZHANG Yigang;FENG Xiaojia;ZHANG Lingmei;JIA Jianguang(Department of Surgical Oncology,First Affi-liated Hospital of Bengbu Medical College,Bengbu 233000,China;Department of Hepatobiliary Surgery,First Affiliated Hospital of Bengbu Medical College;Department of Gynecological Oncology,First Affiliated Hospital of Bengbu Medical College)

机构地区：[1]蚌埠医学院第一附属医院肿瘤外科,蚌埠233000 [2]蚌埠医学院第一附属医院肝胆外科,蚌埠233000 [3]蚌埠医学院第一附属医院肿瘤妇科,蚌埠233000

出　　处：《山西医科大学学报》2021年第11期1379-1388,共10页Journal of Shanxi Medical University

基　　金：蚌埠医学院512人才培育计划项目(by51202207);蚌埠医学院研究生科研创新计划项目(Byycx20042)。

摘　　要：目的探讨SMC4基因在贲门腺癌细胞SGC-7901中发挥的作用及内在机制。方法收集20例贲门腺癌患者的贲门腺癌组织及贲门腺癌癌旁正常组织,送检进行蛋白质谱检测分析,获得差异表达基因SMC4。并经Western blot测定SMC4在贲门腺癌细胞MKN-45、BGC-823、SGC-7901、MGC-803和正常胃上皮细胞GES-1中的蛋白水平。随后选择SMC4表达量适中的SGC-7901细胞构建后续实验对象,实验分为NC组(共转染polybrene和对照shRNA重组慢病毒液)、SMC4-RNAi1组(共转染polybrene和SMC4 shRNA-1重组慢病毒液)和SMC4-RNAi2组(共转染polybrene和SMC4 shRNA-2重组慢病毒液)。运用MTT试验和克隆形成试验评估敲除SMC4对细胞增殖活性的影响,通过划痕试验、Transwell试验研究下调SMC4对细胞迁移、侵袭的作用,使用流式细胞术检测SMC4对细胞周期的影响,通过Western blot分析SMC4对凋亡相关蛋白(Caspase-3、Bcl-2、cleaved Caspase-3、Bax)和周期相关蛋白(CDK6、Cyclin D1、P21、CDK4)的影响,进行Western blot实验以确定SMC4对上皮-间质转化(EMT)关键蛋白(Vimentin、N-cadherin、E-cadherin)和PI3K/AKT信号通路相关蛋白(磷酸化mTOR、磷酸化AKT、磷酸化PI3K)的调控作用。结果蛋白质谱分析结果显示,贲门腺癌组织中SMC4的蛋白表达量显著高于癌旁组织(P<0.05)。与正常胃上皮细胞进行对比,SMC4在贲门腺癌细胞中的蛋白表达水平较高(P<0.05)。与NC组比较,SMC4-RNAi1组和SMC4-RNAi2组SMC4蛋白表达量显著减少(P<0.05)。与NC组比较,SMC4-RNAi1组和SMC4-RNAi2组SGC-7901细胞的侵袭、增殖、迁移活性降低(P<0.05)。与NC组相比,SMC4-RNAi1组和SMC4-RNAi2组G0/G1期细胞显著增加,而S期和G2/M期细胞数则减少(P<0.05)。此外,SMC4-RNAi1组和SMC4-RNAi2组的Vimentin、CDK6、Bcl-2、Cyclin D1、N-cadherin及CDK4的蛋白水平相较于NC组降低,而Bax、Caspase-3、cleaved Caspase-3、P21和E-cadherin蛋白水平升高(P<0.05)。与NC组相比,SMC4-RNAi1组和SMC4-RNAi2组磷Objective To explore the role of SMC4 gene in cardia adenocarcinoma SGC-7901 cell and its mechanism.Methods The cardia adenocarcinoma tissues and the normal tissues adjacent to cardia adenocarcinoma were collected from 20 patients with cardia adenocarcinoma.Protein spectrum analysis was performed to obtain the differentially expressed gene SMC4.The protein level of SMC4 in cardia adenocarcinoma cells MKN-45,BGC-823,SGC-7901,MGC-803 and normal gastric epithelial cells GES-1 was detected by Western blot.Subsequently,SGC-7901 cells with moderate SMC4 expression were selected to construct subsequent experimental subjects.The cells were divided into NC group(co-transfected with polybrene and control shRNA recombinant lentivirus),SMC4-RNAi1 group(co-transfected with polybrene and SMC4 shRNA-1 recombinant lentivirus)and SMC4-RNAi2 group(co-transfected with polybrene and SMC4 shRNA-2 recombinant lentivirus).The effects of SMC4 knockdown on cell proliferation were detected by MTT assay and clone formation assay.The effects of SMC4 downregulation on cell migration and invasion were studied by scratch assay and Transwell experiments.The effect of SMC4 on cell cycle was detected by flow cytometry.The effects of SMC4 on apoptosis-related proteins(Caspase-3,Bcl-2,cleaved Caspase-3,Bax)and cycle-related proteins(CDK6,Cyclin D1,P21,CDK4)were detected by western blot.The regulation of SMC4 on key EMT proteins(Vimentin,N-cadherin,E-cadherin)and PI3K/AKT signaling pathway related proteins(phosphorylated mTOR,phosphorylated AKT,and phosphorylated PI3K)was observed by Western blot.Results Protein spectrum analysis showed that the protein level of SMC4 in cardia adenocarcinoma tissues was significantly higher than that in paracancer tissues(P<0.05).Compared with normal gastric epithelial cells,the protein level of SMC4 was increased in cardia adenocarcinoma cells(P<0.05).Compared with NC group,the protein expression of SMC4 in SMC4-RNAi1 and SMC4-RNAi2 group was significantly dropped off(P<0.05).Compared with NC group,the capacities o

关 键 词：SMC4 PI3K/AKT信号通路 贲门腺癌 

分 类 号：R735.2[医药卫生—肿瘤]