S100A4在食管鳞癌上皮间质转化过程中的作用及其分子机制

Role of S100A4 in the epithelial-mesenchymal transition of esophageal squamous cell carcinoma and its molecular mechanism

目的探讨钙结合蛋白S100A4在食管鳞癌上皮间质转化（EMT）过程中的作用及其可能的分子机制。方法化学合成靶向S100A4的3对siRNA序列瞬时转染EC9706细胞，同时以转染阴性对照siRNA、脂质体和空白EC9706细胞作为对照，荧光定量RT—PCR和Westernblot法检测其抑制效率。将抑制效果最佳的S100A4siRNA2以相同条件转染食管癌EC9706细胞，同时以转染阴性对照siRNA、脂质体和空白EC9706细胞作为对照；然后在转染S100A4siRNA2的EC9706细胞中转染snarl真核表达载体，并以S100A4siRNA2组、snail真核表达载体组和空白组为对照，分别采用荧光定量RT—PCR和Westernblot法检测E—cadherin、vimentin和snail的表达，倒置显微镜观察各组EC9706细胞形态变化，Boydenchamber和划痕实验检测各组EC9706细胞侵袭迁移能力的变化，CCK-8法检测各组EC9706细胞增殖能力的变化。结果S100A4siRNA2组S100A4mRNA和蛋白的相对表达量分别为0．417±0．041和0．337±0．039，穿膜细胞数为（61．608±8．937）个，划痕愈合距离为（0．216±0．064）mm，A值为0．623±0．084，E—cadherinmRNA和蛋白的相对表达量分别为0．619±0．032和0．495±0．034，vimentin mRNA和蛋白的相对表达量分别为0．514±0．032和0．427±0．028，snailmRNA和蛋白的相对表达量分别为0．573±0．029和0．429±0．041，与脂质体组、阴性对照组和空白组比较，差异均有统计学意义（均P〈0．05）。S100A4siRNA2作用于EC9706细胞24h后，细胞形态向上皮细胞形态转变，呈去梭形化趋势。S100A4siRNA2＋snail真核表达载体组的穿膜细胞数和划痕愈合距离分别为（69．382±9．666）个和（0．274±0．029）mm，A值为0．823±0．101，snailmRNA和蛋白的相对表达量分别为0．704±0．037和0．625±0．031，vimentinmRNA和蛋白的相对表达量分别为0．712±0．046和0．609±0．038，高于S100A4siRNA2组（均P〈0．05）；S100A4siRNA2＋snail真核表达载体组中E—cadhefinmRNA和蛋白的相对表达量分别为0．437±0．038和0．381±0．031，低于S100A4siRNA2组（均P〈0．05）。但snail表达增高对EC9706细胞形态无影响。结论S100A4可通过调控snail的表达参与食管癌EMT过程，进而在食管癌浸润转移中发挥作用。

Objective To explore the role of S100A4 in the epithelial-mesenchymal transition （EMT） in esophageal squamous cell carcinoma and its possible molecular mechanism. Methods Three chemically synthesized S100A4 siRNA sequences were transiently transfected into esophageal carcinoma EC9706 cells. EC9706 cells transfected with negative siRNA, lipofeetamine 2000, and vacant EC9706 cells were used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the inhibition rate of S100A4 siRNA. SIOOA4 siRNA2 with the best inhibition rate was chosen to transiently transfect into EC9706 cells under the same conditions. The EC9706 cells transfected with negative siRNA, lipofectamine 2000 and vacant EC9706 cells were also used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the mRNA and protein expressions of E-cadherin, vimentin and snail. The morphology of EC9706 cells was observed under an inverted microscope. Boyden chamber and scratch test were used to detect the invasion and migration ability of EC9706 cells, and CCK8 assay was used to detect the proliferation ability of EC9706 cells. EC9706 cells transfected with S100A4 siRNA2 were further transfected with snail eukaryotic expression vector. The EC9706 cells transfected with S100A4 siRNA, EC9706 cells transfected with snail eukaryotic expression vector and vacant EC9706 cells were used as control. The above indexes of all the groups were observed, too. Results The S100A4 mRNA and protein expression levels of the S100A4 siRNA2 group were 0.417±0.041 and 0.337±0.039, the transmembrane cell number was 61.608±8.937, the scratch healing distance was （0.216＋0.064） mm, the A value was 0.623±0.084, the E-cadherin mRNA and protein levels were 0.619±0.032 and 0.495±0.034, the vimentin mRNA and protein levels were 0.514±0.032 and 0.427±0.028, the snail mRNA and protein levels were 0.573±0.029 and 0.429±0.041. These data were significantly different with the liposome group, the negative control group and the blank group （P〈0.05 for all）. After the S100A4 siRNA2 treatment for 24 h, the appearance of EC9706 cells changed to epithelial cell morphology. The transmembrane cell number and the scratch healing distance of the S100A4 siRNA2＋snail eukaryotic expression vector group were （69.382±9.666） cells and （0.274±0.029） mm, the A value was 0.823±0.101, the snail mRNA and protein levels were 0.704±0.037 and 0.625±0.031, the vimentin mRNA and protein levels were 0.712±0.046 and 0.609±0.038, and these data were significantly higher than those of the SIOOA4 siRNA2 group （P〈0.05 for all）. The E-cadherin mRNA and protein levels of the S100A4 siRNA2 ＋ eukaryotic expression vector group were 0.437 ±0.038 and 0.381 ±0.031, significantly lower than those of the S100A4 siRNA2 group （P〈0.05 for all）. However, snail had no effect on the morphology of EC9706 ceils. Conclusions S100A4 may be involved in the EMT process of esophageal squamous-cell carcinoma by regulating the expression of snail and then plays a role in the invasion and metastasis of esophageal carcinoma.