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.
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。
Keywords: GEPIA database; TIM-3 gene; Wnt/β-catenin signaling pathway; biological behaviors; epithelial ovarian cancer.