S100A4真核表达载体构建及对鼻咽癌细胞侵袭转移的影响

Establishment of S100A4 eukaryotic expression vector and the effects to metastasis and invasion of NPC

目的构建S100A4稳定高表达的鼻咽癌细胞株,研究S100A4与鼻咽癌侵袭转移关系。方法 PCR方法扩增S100A4片段,将产物插入p CMV载体,将重组质粒及其空白质粒分别转染到鼻咽癌细胞CNE2;MTT法绘制细胞生长曲线,Western blot检测S100A4、E-cadherin、Fibronectin、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)的表达;Transwell实验检测细胞侵袭转移能力变化。结果成功构建了S100A4稳定高表达的重组细胞株;细胞生长曲线表明S100A4稳定高表达的重组细胞株生长较快;S100A4稳定高表达的重组细胞株中E-cadherin、Fibronectin低表达而MMP2、MMP9高表达;Transwell实验发现S100A4稳定高表达的重组细胞株的侵袭细胞计数明显高于空白载体转染组和未转染组(P>0.01)。结论 S100A4能调控鼻咽癌上皮-间质转化(EMT)过程,并上调MMP2、MMP9表达而促进鼻咽癌细胞侵袭和转移。

Objective To build a stable cell line with S100A4 high expression and study the relationship between S100A4 and metastasis and invasion of nasopharyngeal carcinoma（NPC）. Methods To amplify the fragment of S100A4 by PCR method,and PCR products were inserted into p CMV vector. The reconstructed p CMV-S100A4 vector and empty vector were transferred into CNE2 cell line. Then the growth curve were drown by MTT method. The expression of S100A4,E-cadherin,Fibronectin,MMP2（matrix metallopeptidase 2） and MMP9（matrix metallopeptidase 9） were detected by Western blot. And the changes of ability of metastasis and invasion were examined by Transwell. Results Succeed to build a stable cell line with S100A4 high expression; The grow curves showed that the growth speed were more quickly in p CMV-S100A4-CNE2 than in p CMV-empty-CNE2 and CNE2. Western blot results showed that there were low expression of E-cadherin、Fibronectin and high expression of MMP2,MMP9 in p CMV-S100A4-CNE2 than in p CMV-empty-CNE2 and CNE2. Transwell results showed that there were more invaded cells in p CMV-S100A4-CNE2 than in p CMV-empty-CNE2 and CNE2（P〈0.01）. Conclusions S100A4 can regulate the process of EMT in NPC and upregulate the expression of MMP2 and MMP9 to promote the metastasis and invasion of NPC cells.