链接蛋白-4在食管癌组织的表达及临床意义

Over-expression of Nectin-4 promotes progression of esophageal cancer and correlates with poor prognosis of the patients

目的观察链接蛋白-4(Nectin-4)在人食管癌组织及邻近正常组织的表达,分析其表达水平与食管癌患者临床病理特征及预后的关系.探讨Nectin-4干预对人食管癌细胞株的功能调节作用.方法采用免疫组织化学染色法检测食管癌组织芯片中94例食管癌和78例相邻癌旁正常组织Nectin-4的表达,采用秩和检验比较食管癌及癌旁正常组织中Nectin-4表达水平的差异,x2检验分析食管癌组织中Nectin-4表达水平与患者临床病理特征的关系,拟合Cox模型评价不同指标与患者预后的关系.选用人食管癌细胞株Eca-109和TE-1,经RNA干扰(RNAi)技术构建Nectin-4稳定下调及过表达细胞株,分别通过实时定量聚合酶链反应(Real-time PCR)和蛋白质印迹法(Western blot)进行验证,通过细胞计数试剂盒(CCK-8)细胞增殖实验、划痕实验及Transwell实验探讨Nectin-4干预对上述食管癌细胞株生物学功能的调节作用.结果Nectin-4在食管癌组织中高表达,在正常组织中以不表达和低表达为主,在癌组织及癌旁组织中的表达差异有统计学意义(P<0.01).肿瘤最大直径≥4.5 cm的食管癌患者其Nectin-4高表达比率高于最大直径<4.5 cm的患者,差异有统计学意义(x^2=6.385,P<0.05).T分期为Ⅲ+Ⅳ的食管癌患者其Nectin-4高表达比率高于TNM分期为Ⅰ+Ⅱ的食管癌患者,差异有统计学意义(x^2=5.822,P<0.05).Nectin-4染色强度与患者其他临床病理特征无相关(P>0.05).多因素Cox比例风险模型显示,Nectin-4表达水平[风险比(HR)=1.795,95%可信区间(CI):1.042~3.092,P<0.05]可作为食管癌患者独立预后因素.经过RNAi及慢病毒转染,选用Eca-109和TE-1成功构建Nectin-4稳定下调表达细胞株LV-Nectin-4-短发卡RNA(shRNA)和Nectin-4过表达细胞株LV-Nectin-4-OE.CCK-8实验显示,LV-Nectin-4-shRNA组细胞的增殖率显著低于LV-NC组细胞(Eca-109,48 h:P<0.01,72 h:P<0.05;TE-1,24h:P<0.01,48 h:P<0.01,72 h:P<0.01),LV-Nectin-4-OE组细胞增殖率显著高于LV-Vector-Ctrl组(Eca-109,48 h:P<0.05,72 h:P<0.05;TE-1,48 h:P<0.01,72 h:P<0.05).划痕实验显示,LV-Nectin-4-shRNA组细胞迁移能力显著低于LV-NC组(Eca-109,24 h:P<0.05;TE-1,24 h:P<0.01);LV-Nectin-4-OE组细胞迁移能力显著高于LV-Vector-Ctrl组(Eca-109,24 h:P<0.01;TE-1,24h:P<0.05).Transwell侵袭实验显示,LV-Nectin-4-shRNA组细胞侵袭数量显著低于LV-NC组(Eca-109,P<0.05;TE-1,P<0.01);LV-Nectin-4-OE组细胞侵袭数量显著高于LV-Vector-Ctrl组(Eca-109,P<0.01;TE-1,P<0.01).皮下移植瘤实验证实,敲除Nectin-4表达可以显著抑制肿瘤生长(Eca-109,P<0.01;TE-1,P<0.01);LV-Nectin-4-shRNA组肿瘤组织重量显著轻于LV-NC组(Eca-109,P<0.05;TE-1,P<0.01);LV-Nectin-4-OE组肿瘤组织重量显著重于LV-Vector-Ctrl组(Eca-109,P<0.018;TE-1,P>0.05).结论Nectin-4在食管癌组织中高表达,表达水平与食管癌患者肿瘤大小、T分期密切相关,Nectin-4高表达可作为食管癌患者预后评判的独立风险因素,提示Nectin-4参与食管癌的发生与进展.人食管癌细胞中下调Nectin-4表达可抑制细胞活力、迁移和侵袭的能力,上调Nectin-4表达可增强细胞活力、迁移和侵袭的能力.

Objective To investigate the expression of Nectin-4 and to analyze its clinical significance.Methods The immunohistochemistry assay and the tissue micro-array were used to examine the Nectin-4 expression in human EC tissues as well as adjacent normal tissues.The correlation between the expression level of Nectin-4and patients’clinic pathological parameters was evaluated by using chi-square test.The human EC cell lines Eca-109 and TE-1 were used to construct the stable knockdown or overexpression of Nectin-4 using RNA interference(RNAi)method.The cell counting kit-8(CCK-8),wound healing,and Transwell were used to examine the cellular function in Nectin-4-knockdown expression or Nectin-4-over-expressing cell lines.In order to study the effect of abnormal Nectin-4 expression on the regulation of tumor growth in vivo,the subcutaneous transplantation mouse model were established.Results The staining intensity of Nectin-4 in EC tissues was significantly higher than that in adjacent normal tissues(P<0.01).The survival analysis showed that the overall survival rate of the patients with higher Nectin-4 expression was significantly poorer compared with those showing lower Nectin-4 expression[hazard ratio(HR)=1.704,95%confidence interval(CI):1.027-2.825,P<0.05].The staining intensity of Nectin-4 was positively and significantly associated with tumor size(χ^2=6.385,P<0.05)and tumor stage(χ^2=5.822,P<0.05).According to the COX model analysis,the Nectin-4 expression level could serve as an independent prognostic predictor for EC patients(HR=1.795,95%CI:1.042-3.092,P<0.05),suggesting that abnormal expression of Nectin-4 was involved in the progression of EC.CCK-8 assay showed that,the proliferation rate of LV-Nectin-4-short hairpin RNA(shRNA)group cells was significantly lower than that of LV-NC group cells(Eca-109,48 h:P<0.01,72 h:P<0.05;TE-1,24 h,P<0.01,48 h:P<0.01,72 h:P<0.01).The proliferation rate of LV-Nectin-4-OE group cells was significantly higher than that of LV-Vector-Ctrl group cells(Eca-109,48 h:P<0.05,72 h:P<0.05;TE-1,48 h:P<0.01,72 h:P<0.05).The wound healing assay showed that the cell migration abilities of LV-Nectin-4-shRNA group was significantly decreased than LV-NC group(Eca-109,24 h:P<0.05;TE-1,24 h:P<0.01).The cell migration abilities of LV-Nectin-4-OE group was significantly increased than LV-Vector-Ctrl group(Eca-109,24 h:P<0.01;TE-1,24 h:P<0.05).The Transwell assay showed that the cell invasion abilities of LV-Nectin-4-shRNA group was significantly lower than that in LV-NC group(Eca-109,P<0.05;TE-1,P<0.01).The cell invasion abilities of LV-Nectin-4-OE group was significantly higher than that in LV-Vector-Ctrl group(Eca-109,P<0.01;TE-1,P<0.01).The subcutaneous transplantation mouse model showed that knockdown expression of Nectin-4 significantly inhibited the tumor growth(Eca-109,P<0.01;TE-1,P<0.012).The tumor weight of the LV-Nectin-4-shRNA group was lighter compared with the LV-NC group(Eca-109,P<0.05;TE-1,P<0.01),and the tumor weight of the LV-Nectin-4-OE group trended to be heavier compared with the LV-Vector-Ctrl group(Eca-109,P<0.018;TE-1,P>0.05).Conclusion The expression of Nectin-4 is higher in in human EC tissues than that in adjacent normal tissues and its expression level was significantly correlated with tumor size and T stage EC patients.The Nectin-4 expression level could serve as an independent prognostic predictor for EC patients,suggesting that abnormal expression of Nectin-4 was involved in the progression of EC.Its knockdown could promote cell proliferation,migration and invasion of cancer cells,and thus could be used as potential therapeutic target for human EC.