结肠癌T-cadherin表达水平分析与5-Aza-CdR对其表达和结肠癌细胞增殖、侵袭及凋亡的影响

Analysis of T-cadherin expression in colon cancer and the effect of 5-Aza-CdR on T-cadherin expression and proliferation,invasion and apoptosis of colon cancer cells

背景与目的:结肠癌是临床常见恶性肿瘤,探讨抑癌蛋白T-cadherin在结肠癌中的表达情况及其与患者临床病理学特征的关系,并分析5-Aza-CdR对T-cadherin表达和结肠癌细胞增殖、侵袭及凋亡的影响。方法:收集福建医科大学附属第二医院2015—2016年40例手术切除的结肠癌组织及癌旁组织新鲜样本,并经过病理学检查验证,分别提取总RNA和总蛋白质。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)分析T-cadherin mRNA的表达,采用蛋白质印迹法(Western blot)分析T-cadherin蛋白水平,并分析T-cadherin的mRNA表达变化与患者临床病理学特征的关系。进一步以人结肠癌细胞系HT-29为研究对象,采用甲基化抑制剂5-Aza-CdR处理HT-29细胞,分别采用RTFQ-PCR和Western blot分析T-cadherin表达变化,采用细胞计数试剂盒(cell counting kit-8,CCK-8)分析细胞增殖,采用Transwell实验验证细胞侵袭能力,采用流式细胞术分析细胞凋亡。结果:T-cadherin在结肠癌组织的蛋白水平和mRNA表达均明显低于癌旁组织,其mRNA表达与淋巴结转移(F=5.316,P=0.0093)和分化程度(F=5.807,P=0.0064)明显相关,而与其他病理变量(包括性别、年龄、肿瘤大小和肿瘤浸润深度)无明显相关。药物5-Aza-CdR可以显著上调HT-29细胞中T-cadherin的表达水平,抑制HT-29细胞增殖、侵袭并促进细胞凋亡。结论:T-cadherin表达可能与结肠癌恶性程度密切相关,药物5-Aza-CdR处理可上调结肠癌细胞T-cadherin的表达,并抑制结肠癌细胞的增殖、迁移,促进结肠癌细胞凋亡,提示T-cadherin可能是结肠癌患者使用甲基化抑制剂5-Aza-CdR治疗的靶点。

Background and purpose:Colon cancer is a common clinical malignant tumor.This study aimed to investigate the expression of tumor suppressor gene expression product T-cadherin in colon cancer and its relationship with clinicopathological features,and to analyze the effect of 5-Aza-CdR on T-cadherin expression and proliferation,invasion and apoptosis of colon cancer cells.Methods:The fresh samples of colon cancer tissues and adjacent tissues from the Second Affiliated Hospital of Fujian Medical University were collected and verified by pathological diagnosis from 2015 to 2016,and total RNA and protein were extracted respectively.The expression of T-cadherin in tissues was analyzed by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot,and the relationship between the expression of T-cadherin and clinicopathological features was also analyzed.Furthermore,colon cancer HT-29 cells were treated with methylation inhibitor 5-Aza-CdR.The expression of T-cadherin in cells was analyzed by RTFQ-PCR and Western blot.Cell proliferation,invasive ability and apoptosis were analyzed by cell counting kit-8(CCK-8),transwell assay and flow cytometry respectively.Results:The T-cadherin expression in colon cancer tissues was significantly lower than that in paracancerous tissues.The expression level of T-cadherin was significantly correlated with lymph node metastasis(F=5.316,P=0.0093)and the degree of differentiation(F=5.807,P=0.0064).Other pathologic parameters,including gender,age,tumor size and depth of tumor invasion,were not significantly correlated with T-cadherin expression.The 5-Aza-CdR treatment significantly up-regulated the expression of T-cadherin,inhibited the proliferation and invasion,and promoted the apoptosis of HT-29 cells.Conclusion:The expression of T-cadherin may be closely related to the malignancy of colon cancer.The 5-Aza-CdR treatment up-regulates the expression of T-cadherin,inhibits the cell proliferation and invasion,and promotes the apoptosis of HT-29 cells.It is suggested that T-cadherin could be an important target for 5-Aza-CdR drug therapy in patients with colon cancer.