ERK1/2抑制对沙门菌侵袭小鼠胃癌细胞后小鼠β-防御素-2表达的影响

Effect of ERK1/2 inhibition on expression of mouse β-defensin-2 in mouse gastric cancer cells after invasion with salmonella

目的探讨ERK1/2抑制对沙门菌侵袭小鼠胃癌(mouse forestomach carcinoma,MFC)细胞后小鼠β-防御素-2(mouseβ-defensin-2,m BD-2)表达的影响及作用机制。方法采用RNA干扰的方法抑制MFC细胞中ERK1/2的表达,并经G418筛选稳定细胞株,经RT-PCR和Western blot法检测ERK1/2基因的抑制效率。用10~3个/ml的沙门菌对干扰组及对照组(未转染)的MFC细胞分别作用4、8及12 h,通过Western blot法检测m BD-2蛋白表达及Rsk^(90)的磷酸化水平。同时采用MTT法检测沙门菌作用两组MFC细胞12、24及48 h时的生长情况。结果RT-PCR和Western blot法检测结果表明,ERK1/2基因抑制效果显著。沙门菌作用MFC细胞后4、8和12 h后,各时间点间干扰组细胞中m BD-2蛋白表达及Rsk^(90)蛋白磷酸化水平差异无统计学意义(P>0.05),且均显著低于各时间点的对照组(P<0.05)。沙门菌作用12和24 h,干扰组细胞的抑制率明显高于对照组(P<0.05),作用48 h时,两组细胞的抑制率差异无统计学意义(P>0.05),细胞几乎全部死亡,且干扰组细胞抑制率的增加速度较快,作用24 h时已达97.7%。结论ERK1/2抑制可明显降低沙门菌侵袭MFC后m BD-2的表达水平,表明ERK1/2可调控m BD-2的表达。

Objective To investigate the effect of ERK1/2 inhibition on expression of mouse β-defensin-2（m BD-2） in mouse gastric cancer cells after invasion with salmonella and the relevant mechanism. Methods The expression of ERK1/2 in mouse gastric cancer cells was inhibited by RNA interference, based on which the cell strains stably expressing ERK1/2 were screened with G418, and determined for inhibition efficacy by RT-PCR and Western blot. The mouse gastric cancer cells in interference and control（untransfected） groups were infected with 103 bacteria/ml salmonella for4, 8 and 12 h, and determined for m BD-2 expression and Rsk90 phosphorylation levels by Western blot. Meanwhile, the growth of cells after invasion with salmonella for 12, 24 and 48 h were determined by MTT assay. Results Both RT-PCR and Western blot showed that ERK1/2 gene was inhibited significantly. However, the m BD-2 expression and Rsk90 phosphorylation levels in interference group at various time points after invasion with salmonella showed no significant difference（P 〈 0. 05）, which were significantly lower than those in control group at the corresponding time points（P 〉0. 05）. The inhibition rates of cell growth in interference group were significantly higher than those in control group（P 〈0. 05） 12 and 24 h, while showed no significant difference 48 h after invasion with salmonella. Almost all the cells died,however, the inhibition rate of cell growth in interference group increased rapidly, which reached 97. 7% 24 h after invasion with salmonella. Conclusion ERK1/2 inhibition decreased the m BD-2 expression level in mouse gastric cancer cells after invasion with salmonella, indicating the regulatory effect of ERK1/2 on m BD-2 expression.