胃癌细胞中乳酸脱氢酶A表达变化及其对EMT的影响

Expression changes of lactate dehydrogenase A in gastric cancer cells and its effect on epithelial-mesenchymal transformation

目的 观察胃癌细胞中乳酸脱氢酶A(LDH-A)的表达变化,并探讨沉默LDH-A表达对胃癌细胞上皮间质转化(EMT)的影响。方法 体外培养胃癌细胞株OCUM-1和正常胃黏膜上皮细胞株RGM-1,采用qRT-PCR法检测细胞中LDH-A mRNA相对表达量,Western blotting法检测细胞中LDH-A蛋白表达情况。将OCUM-1细胞随机分为3组,LDH-AsiRNA组采用Lipfoctamine2000脂质体转染试剂对细胞转染LDH-A siRNA序列,转染对照组转染siRNA无义对照序列,空白对照组只加入培养基。转染48h后收集各组细胞,显微镜下观察胃癌细胞的形态学变化,采用Western blotting法检测细胞中上皮细胞特征蛋白上皮性钙黏蛋白(E-cadherin)、细胞角蛋白(Cytokeratin)和间质细胞特征蛋白神经性钙黏蛋白(N-cadherin)、锌指转录因子(Snail)、β-连环蛋白(β-catenin)、波形蛋白(Vimentin)的表达。结果 OCUM-1细胞中LDH-A mRNA及蛋白表达高于RGM-1细胞(P均<0.05)。LDH-AsiRNA组LDH-AmRNA及蛋白表达低于转染对照组和空白对照组(P均<0.05)。显微镜下可见LDH-A siRNA组细胞排列紧密,细胞黏附性增加,表现为上皮细胞特性。LDH-AsiRNA组E-cadherin、Cytokeratin蛋白表达水平高于转染对照组和空白对照组,N-cadherin、Snail、β-catenin、Vimentin蛋白表达水平低于转染对照组和空白对照组(P均<0.05)。结论 胃癌细胞中LDH-A表达升高;沉默LDH-A表达可上调胃癌细胞上皮细胞特征蛋白表达、下调间质细胞特征蛋白表达,从而抑制胃癌细胞的EMT进程。

Objective To observe the expression changes of lactate dehydrogenase A (LDH-A) in gastric cancer cells and to explore the effect of silencing LDH-A expression on epithelial mesenchymal transformation (EMT) in gastric cancer cells. Methods The gastric cancer cell line OCUM-1 and normal gastric mucosal epithelial cell line RGM-1 were cultured in vitro, the relative expression of LDH-A mRNA in cells was detected by qRT-PCR, the expression of LDH-A protein in cells was detected by Western blotting. OCUM- 1 cells were randomly divided into 3 groups, LDH-A siRNA group was transfected with LDH-A siRNA sequence using Lipfoctamine2000 liposome transfection reagent, the transfection control group was transfected with LDH-A siRNANC sequence, and the blank control group was only added with the culture medium. After 48-hour transfection, the cells of each group were collected and the morphological changes of gastric cancer cells were observed under microscope, the expression of E-cadherin, Cytokeratin, N-cadherin, Snail,β-catenin, and Vimentin was detected by Western blotting. Results The mRNA and protein expression of LDH-A in the OCUM-1 cells was higher than that in RGM-1 cells (both P <0.05). The mRNA and protein expression of LDH-A in the LDH-A siRNA group was lower than that in the transfection control group and blank control group (all P <0.05). Under microscopy, the cells in the LDH-A siRNA group were closely arranged, and the cell adhesion increased, showing the characteristics of epithelial cells. The protein expression levels of E-cadherin and Cytokeratin in the LDH-A siRNA group were higher than those in the transfection control group and blank control group, the protein expression levels of N-cadherin, Snail,β-catenin, and Vimentin in the LDH-A siRNA group were lower than those in the transfection control group and blank control group (all P <0.05). Conclusion The expression of LDH-A increases in the gastric cancer cells, and silencing LDH-A expression can up-regulate the expression of epithelial characteristic protein, down-regulate the expression of stromal characteristic protein in gastric cancer cells, and thus inhibit the EMT process of gastric cancer cells.