STAT3与E-cadherin在食管鳞癌上皮间质转化中的作用及机制研究

Study on the role and mechanism of STAT3 and E-cadherin in epithelial mesenchymal transition of esophageal squamous cell carcinoma

目的 探究STAT3与E-cadherin在食管鳞癌中的表达及临床病理相关性,以及在食管鳞癌上皮间质转化中过程中产生的影响.方法 采用RT-qPCR方法检测50例食管鳞癌患者癌组织标本及对应癌旁组织标本中STAT3与E-cadherin的表达;以癌/癌旁正常组织相对表达量表示STAT3与E-cadherin在mRNA水平表达的差异,分析其与患者临床病理特征间的关系;采用简单随机法抽取其中4例食管鳞癌患者组织标本进行Western blotting检测STAT3与E-cadherin的表达情况.采用RT-qPCR及Western blotting方法分别检测3株食管鳞癌细胞株中STAT3与E-cadherin表达,筛选其中高表达的1株进行转染,利用STAT3小干扰RNA作为实验组,以双链无义RNA转染作为对照组,检测STAT3、E-cadherin表达水平变化,并通过细胞划痕实验及Transwell细胞侵袭实验检测实验组细胞迁移与侵袭情况.计量资料以均数±标准差((x)±s)表示,组间比较应用t检验.结果 50例食管鳞癌组织中STAT3相对表达量为1.87 ±0.71,E-cadherin相对表达量为0.63 ±0.28,癌旁组织STAT3为0.56 ±0.45,E-cadhenn为2.01 ±0.68;临床病理特征分析显示,食管鳞癌中STAT3相对高表达、E-cadherin相对低表达程度均与癌组织浸润深度、淋巴结转移与否相关(P＜0.05).其中抽取的4例患者,癌组织中STAT3表达比对应癌旁正常组织高,分别为0.28±0.06、0.05±0.02,P＜0.05,而E-cadherin在癌组织中的表达明显低于在对应癌旁正常组织,分别为0.27±0.07、0.59 ±0.09,P＜0.05.选取高表达的EC109作为实验用食管鳞癌细胞株,RT-qPCR结果显示,STAT3小干扰RNA转染组较其对照组中STAT3表达明显减少,分别为0.85±0.24、3.01 ±0.20,P＜0.05,同时E-cadherin蛋白表达上调,分别为2.69 ±0.29、0.36±0.16,P＜0.05;Western blotting检测显示,相对于对照组,转染STAT3小于扰RNA片段成功抑制STAT3蛋白表达后(分别为0.09±0.04、0.32 ±0.18,P＜0.05),EC109细胞中E-cadherin表达明显上调(分别为0.75 ±0.11、0.15 ±0.05,P＜0.05).下调STAT3表达后,细胞划痕实验及Transwell细胞侵袭实验结果显示,食管鳞癌细胞株EC109中STAT3小干扰RNA转染组迁移率、侵袭细胞数与对照转染双链无义RNA组对比均下降,故下调STAT3表达后迁移、侵袭能力均减弱(P＜0.05).结论 在食管鳞癌中,STAT3转录因子可以通过介导E-cadherin表达,影响上皮间质转化表型,从而影响肿瘤迁移与侵袭.

Objective To investigate the expression and clinical pathological correlation of STAT3 and E-cadherin in esophageal squamous cell carcinoma,as well as the effect of the process in the transformation of epithelial mesenchymal transition of esophageal squamous cell carcinoma.Methods The 50 cases of esophageal squamous cell carcinoma tissues and corresponding adjacent tissues,to detect the expression of STAT3 and E-cadherin expression by RT-qPCR method in carcinoma and adjacent normal tissue expression that differential expression in mRNA level,analyzed its relationship with clinical pathological characteristics between patients;the expression of STAT3 and E-cadherin was detected by Western blotting in 4 cases of esophageal squamous cell carcinoma by simple random sampling.The author detected the expression of STAT3 and E-cadherin in three strains of esophageal squamous cell carcinoma cell lines using RT-qPCR and Western blotting,in which a high expression strains were transfected by STAT3 small interfering RNA screening,as the experimental group,using double stranded nonsense RNA transfection as the control group,detected the changes of expression level of STAT3 and E-cadherin.Detected cell migration and invasion by cell scratch test and Transwell cell invasion test in the experimental group.Measurement data were expressed by ((x) ± s),and the t test was used comparison between groups.Results In 50 cases of esophageal carcinoma,the relative expression of STAT3 was 1.87 ± 0.71,E-cadherin was 0.63 ± 0.28,in paracancerous tissue STAT3 was 0.56 ± 0.45,E-cadherin was 2.01 ± 0.68;analysis of clinical pathological features of esophageal squamous cell carcinoma showed relatively high expression of E-cadherin as relatively low expression levels of STAT3,related to the depth and lymph node metastasis in carcinoma tissues (P ＜ 0.05).Among which 4 cases were selected,the expression of STAT3 in cancer tissues higher than in corresponding adjacent normal tissues,0.28 ± 0.06 vs 0.05 ± 0.02,P ＜ 0.05,and the expression of E-cadherin in cancer tissues was significantly lower than that in adjacent normal tissues,0.27 ± 0.07,0.59 ± 0.09,P ＜ 0.05.The high expression of EC109 from carcinoma cell lines was as experimentation of esophageal squamous cell,RT-qPCR results showed that the expression of STAT3 was significantly decreased in small interfering RNA transfection group than the control group 0.85 ± 0.24 vs 3.01 ± 0.20,P ＜ 0.05,while up-regulation of E-cadherin protein expression,2.69 ± 0.29 vs 0.36 ± 0.16,P ＜ 0.05;Western blotting detection,compared with the control group,the expression of STAT3 protein was successfully inhibited after transfection of STAT3 small interference RNA fragment (0.09 ±0.04 vs 0.32 ±0.18,P ＜ 0.05),while the expression of E-cadherin was up-regulated in EC109 cells (0.75 ±0.11 vs 0.15 ±0.05,P ＜0.05).After downregulation of STAT3 expression,cell scratch test and transwell cell invasion test showed that the migration and the number ofinvasion cell number of STAT3 small interfering RNA transfection group in esophageal squamous carcinoma cell line EC109 compared with the control transfected double-stranded non-sense RNA group were decreased,so the downregulation of STAT3 expression after migration,invasion ability were weakened (P ＜ 0.05).Conclusion In esophageal squamous cell carcinoma,STAT3 transcription factor can mediate E-cadherin expression and affect epithelial mesenchymal transition phenotype,thereby affecting tumor migration and invasion.