NET-1通过激活EMT过程促进肺鳞癌转移

NET-1 promotes metastasis of lung squamous-cell carcinoma by activating EMT

目的:探讨神经上皮细胞转化基因1(NET-1)表达对肺鳞癌(LSC)转移的影响及分子机制。方法:免疫组化检测53例肺鳞癌(LSC组)、24例正常肺上皮(NLE组)及27例肺鳞状上皮内病变(SIL组)组织中NET-1蛋白的表达,并统计分析其表达水平与临床病理因素的相关性;Western blot实验检测人肺鳞癌细胞系H226、H1703、H2170、SK-MES-1、H520和YTMLC-90中NET-1的表达;构建针对NET-1基因的RNA干扰重组腺病毒(Ad-NET-siRNA)及含对照序列的重组腺病毒(Ad-control)感染人肺鳞癌细胞YTMLC-90;Western blot实验检测人正常肺上皮细胞BEAS-2B及人肺鳞癌细胞YTMLC-90中各组NET-1、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)蛋白和Snail1蛋白的表达变化;通过qPCR实验检测各组细胞E-cadherin和vimentin的mRNA表达;利用Transwell小室侵袭实验检测各组细胞侵袭能力的变化。结果:LSC组中NET-1的阳性表达率显著高于NLE组和SIL组(P<0.05)。NET-1蛋白阳性表达人群的分布与组织学分级、是否有淋巴结转移以及TNM分期相关,其中淋巴结有转移组的NET-1表达率显著高于淋巴结无转移组。体外实验发现,YTMLC-90细胞中的NET-1蛋白过表达并促进细胞侵袭,并且在YTMLC-90细胞中E-cadherin的mRNA和蛋白表达降低,vimentin的mRNA和蛋白以及Snail1蛋白表达升高;而随着敲减NET-1的表达,YTMLC-90细胞中E-cadherin的mRNA和蛋白表达升高,vimentin的mRNA和蛋白以及Snail1蛋白表达降低。结论:NET-1表达可促进肺鳞癌淋巴转移,这种促进作用可能是通过表达NET-1从而激活上皮-间充质转化(EMT)过程实现的。

AIM: To explore the effect of neuroepithelial cell transforming gene-1(NET-1) expression on the metastasis of lung squamous-cell carcinoma(LSC) and the underlying molecular mechanism. METHODS: Immunohistochemistry was used to detect the expression of NET-1 protein in 53 cases of lung squamous-cell carcinoma(LSC group), 24 cases of normal lung epithelium(NLE group) and 27 cases of lung squamous intraepithelial lesions(SIL group). The correlation of clinical and pathological factors was analyzed. The protein expression of NET-1 in human lung squamous-cell carcinoma cell lines H226, H1703, H2170, SK-MES-1, H520 and YTMLC-90 was determined by Western blot. The RNA interference recombinant adenovirus against NET-1 gene(Ad-NET-siRNA) and Ad-control with control sequence were constructed and infected with human lung squamous cell carcinoma cell YTMLC-90 to silence the expression of NET-1 gene. The protein expression of NET-1, E-cadherin, vimentin and Snail1 in the BEAS-2 B cells and the YTMLC-90 cells was determined by Western blot. The mRNA expression of E-cadherin and vimentin in each group of the cells was detected by qPCR. The invasive ability of the cells in each group was detected by Transwell chamber assay. RESULTS: The positive expression rate of NET-1 in LSC group was significantly higher than that in NLE group and SIL group(P<0.05). The distribution of NET-1 protein positive expression population was correlated with histological grade, lymph node metastasis, and TNM stage. The NET-1 expression rate of LSC with lymph node metastasis was significantly higher than that without lymph node metastasis. Over-expression of NET-1 protein in YTMLC-90 cells was observed. The expression of E-cadherin was decreased, and the protein expression of vimentin and Snail1 was increased in YTMLC-90 cells. Knock-down of NET-1 expression increased the expression of E-cadherin, and decreased the expression of vimentin and Snail1 in the YTMLC-90 cells. CONCLUSION: The expression of NET-1 promotes the lymphatic metastasis of lung squamous-cell carcinoma. This promotion may be achieved through the activation of epithelial-mesenchymal transition(EMT) by NET-1 expression.