TIM-3基因在上皮性卵巢癌中高表达并促进癌细胞的增殖和迁移  

TIM 3 gene is highly expressed in ephithelial ovarian cancer to promote proliferation and migration of ovarian cancer cells

作　　者：霍叶琳 王月[3] 安娜[3] 杜雪[1] HUO Yelin;WANG Yue;AN Na;DU Xue(Department of Gynecology,General Hospital of Tianjin Medical University,Tianjin 300000,China;Department of Gynecology,Baoding First Hospital,Baoding 071000,China;Department of Oncology,Affiliated Hospital of Hebei University,Baoding 071000,China)

机构地区：[1]天津医科大学总医院妇科,天津300000 [2]河北省保定市第一医院妇科,河北保定071000 [3]河北大学附属医院肿瘤内科,河北保定071000

出　　处：《南方医科大学学报》2022年第2期190-200,共11页Journal of Southern Medical University

摘　　要：目的通过挖掘GEPIA数据库中的基因信息,分析T细胞免疫球蛋白黏蛋白分子3(TIM-3)基因在上皮性卵巢癌(EOC)中的表达及作用机制。方法采用GEPIA数据库在线分析TIM-3基因在EOC组织和正常卵巢组织中的表达水平。收集2018年6月~2019年12月在我院实施卵巢手术治疗的82例EOC癌变组织标本和18例正常卵巢组织标本,免疫组织化学法检测标本组织中TIM-3的表达水平,分析TIM-3表达与EOC临床病理参数的相关性;采用Kaplan-MeierPlotter分析TIM-3表达水平与EOC患者生存之间的关系。qRT-PCR法检测卵巢癌组织和正常组织标本TIM-3和Wnt1 mRNA表达水平及相关性。采用pMAGic 4.0质粒转染构建TIM-3沉默卵巢癌SKOV3细胞系,将细胞分为SKOV3组(未转染的SKOV3细胞系)、沉默阴性对照组(SKOV3+NCsiRNA组:转染pMAGic 4.0 NC siRNA质粒的SKOV3细胞系)和沉默TIM-3组(SKOV3+TIM-3 siRNA组:转染pMAGic 4.0 TIM-3 siRNA质粒的SKOV3细胞系);采用pcDNA3.1质粒转染构建TIM-3过表达卵巢癌SKOV3细胞系,将细胞分为SKOV3组、过表达阴性对照组(pcDNA NC组:转染pcDNA3.1质粒的SKOV3细胞系)和过表达TIM-3组(pcDNA TIM-3组:转染pcDNA3.1 TIM-3质粒的SKOV3细胞系)。MTT法检测细胞增殖能力,Annexin V-FITC/PI染色检测细胞凋亡能力,划痕实验和Transwell实验检测细胞迁移和侵袭能力,TOPflash/FOPflash双荧光素酶报告基因实验检测Wnt/β-catenin通路活性,qPCR检测Wnt/β-catenin信号通路中转录因子TCF-7、TCFL-2及靶基因CD44的mRNA水平,Westernblot检测SKOV3细胞中MMP-9,CD44、Wnt1、β-catenin及E-cad蛋白表达。结果TIM-3在正常卵巢组织中呈阴性表达,在EOC组织中呈阳性表达,EOC组织中TIM-3阳性表达率(84.14%)显著高于正常卵巢组织(16.67%,P<0.05)。TIM-3表达与FIGO分期、组织分化程度及淋巴结是否转移有关(P<0.05),与年龄、病理类型无关(P>0.05)。且TIM-3与Wnt1水平呈正相关(P<0.05)。沉默TIM-3基因后,Wnt/β-catenin通路活性受到抑�Objective To analyze the expression of immunoglobulin mucin molecule 3(TIM-3)in epithelial ovarian cancer(EOC)and the effects of TIM-3 knockdown and overexpression on proliferation and migration of ovarian cancer cells.Methods We analyzed TIM-3 expression in EOC and normal ovarian tissues using GEPIA database.We also detected TIM-3 expression levels in 82 surgical specimens of EOC and 18 specimens of normal ovarian tissues using immunohistochemistry,and analyzed the correlation of TIM-3 expression with clinicopathological parameters and survival outcomes of the patients.The expression of TIM-3 and Wnt1 mRNA in the tissues were detected using qRT-PCR.We constructed SKOV3 cell models of TIM-3 knockdown and overexpression and examined the changes in proliferation,apoptosis,migration and invasion of the cells using MTT assay,Annexin V-FITC/PI staining,scratch test and Transwell assay.The activity of Wnt/β-catenin pathway in the transfected was detected using dual luciferase reporter assay,and the mRNA levels of TCF-7,TCCFL-2 and CD44 were detected using qPCR.The protein expressions of MMP-9,CD44,Wnt1,β-catenin and E-cad in the transfected cells were detected with Western blotting.Results The positive expression rate of TIM-3 was significantly higher in EOC tissues than in normal ovarian tissues(P<0.05).The expression of TIM-3 was significantly correlated with FIGO stage,histological differentiation and lymph node metastasis,and was positively correlated with Wnt1 level(P<0.05).In SKOV3 cells,TIM-3 knockdown significantly lowered the activity of Wnt/β-catenin pathway,inhibited cell proliferation,migration and invasion,and promoted cell apoptosis.TIM-3 knockdown significantly down-regulated the mRNA levels of TCF-7,TCFL-2 and CD44 and the protein levels of MMP-9,CD44,Wnt1 andβ-catenin,and significantly up-regulated the expression level of E-cad(P<0.05).Overexpression of TIM-3 caused opposite effects in SKOV3 cells.Conclusion TIM-3 is highly expressed in EOC tissue to promote malignant behaviors of the tumor cells p

关 键 词：GEPIA数据库 上皮性卵巢癌 TIM-3基因 生物学行为 WNT/Β-CATENIN信号通路 

分 类 号：R737.31[医药卫生—肿瘤]