NK4基因转染对人乳腺癌细胞MDA-MB-231生物学特性的影响

Inhibition of NK4 transfection on biological features of breast cancer cell MDA-MB-231

目的通过在人乳腺癌细胞MDA-MB-231中转染携带有NK4基因的慢病毒载体,研究NK4对乳腺癌细胞的生物学特性影响及其作用机制。方法将NK4重组慢病毒载体pLenti6.3-NK4-IRES-EGFP转染人乳腺癌细胞MDA-MB-231。采用逆转录聚合酶链反应(RT-PCR)及免疫印迹法(Western blot)检测转染细胞中NK4的基因和蛋白表达,检测在肝细胞生长因子(hepatocyte growth factor,HGF)存在的条件下磷酸化c-met受体(p-met)的表达。采用MTT、Transwell小室检测转染NK4基因对乳腺癌细胞的生长、侵袭的影响。结果转染NK4基因的乳腺癌细胞MDA-MB-231能够表达并分泌NK4,RT-PCR扩增出预期的453 bp片段,Western blot显示有50 kD的蛋白并且转染组与空载体组和对照组相比p-met的表达明显减弱。MTT结果显示转染NK4组细胞生长抑制率达(67±2.04)%,较其他两组抑制率高(P<0.01),Transwell结果显示转染NK4基因能够显著抑制由HGF诱导的乳腺癌细胞的侵袭(P<0.01)。结论 NK4通过竞争性抑制HGF诱导的乳腺癌细胞中c-met受体的磷酸化从而抑制了乳腺癌的生长、侵袭。

Objective To investigate the effects of NK4 transfection on biological features of breast cancer cells MDA-MB-231 by transfecting pLenti6.3-NK4-IRES-EGFP into cells.Methods The pLenti6.3-NK4-IRES-EGFP was trasfected into MDA-MB-231 cells.Expression of NK4 gene and protein was identified by RT-PCR and Western blot respectively.Phospho-met（P-met） expression was examined using immunoblotting analysis in the presence of HGF in MDA-MB-231 cells.The effects of NK4 transfection on growth and invasion were detected by MTT assay and Transwell assay.Results The pLenti6.3-NK4-IRES-EGFP transfected MDA-MB-231 cells expressed NK4,and the cells expressed less p-met in the presence of HGF.MTT results showed that the cell number decreased significantly after co-effected by pLenti6.3-NK4-IRES-EGFP and HGF（P〈0.01）.By Transwell assay,pLenti6.3-NK4-IRES-EGFP plus HGF inhibited significantly cell invasion（P〈0.01）.Conclusion In pLenti6.3-NK4-IRES-EGFP trasfected MDA-MB-231 cells,NK4 competitively inhibits the induction of HGF on phosphorylation of c-met.