南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (2): 190-200.doi: 10.12122/j.issn.1673-4254.2022.02.04

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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

霍叶琳，王 月，安 娜，杜 雪   

  1. 天津医科大学总医院妇科，天津 300000；河北省保定市第一医院妇科，河北 保定 071000；河北大学附属医院肿瘤内科，河北 保定 071000
  • 出版日期:2022-02-20 发布日期:2022-03-16

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

HUO Yelin, WANG Yue, AN Na, DU Xue   

  1. 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
  • Online:2022-02-20 Published:2022-03-16

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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-Meier Plotter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+NC siRNA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，Western blot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通路活性受到抑制，SKOV3细胞增殖、迁移和侵袭能力显著降低（P<0.05），凋亡能力显著升高（P<0.05），转录因子TCF-7、TCFL-2及靶基因CD44的mRNA水平显著下调，细胞MMP-9，CD44、Wnt1、β-catenin蛋白水平均显著下调，EMT相关蛋白E-cad水平显著上调（P<0.05）。过表达TIM-3基因后，Wnt/β-catenin通路活性被激活，SKOV3细胞增殖、迁移和侵袭能力显著升高（P<0.05），凋亡能力显著降低（P<0.05），转录因子TCF-7、TCFL-2及靶基因CD44的mRNA水平显著上调，细胞MMP-9，CD44、Wnt1、β-catenin蛋白水平均显著上调，EMT相关蛋白E-cad水平显著下调（P<0.05）。结论 TIM-3在EOC组织中高表达，可促进卵巢癌细胞的恶性生物学行为，其机制可能与Wnt/β-catenin信号通路的激活有关。

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

Abstract: 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 and β-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 possibly by activating the Wnt/β-catenin signal pathway

Key words: GEPIA database; epithelial ovarian cancer; TIM-3 gene; biological behaviors; Wnt/β-catenin signaling pathway