RNA干扰敲减FucT Ⅶ表达抑制人结肠癌细胞HT-29和HUVECs的粘附能力

Knock-down of FucT Ⅶ Expression with RNAi Inhibits the Adhesion of Human Colon Carcinoma HT-29 Cells to HUVEC Cells

探讨利用RNA干扰(RNAi)技术抑制岩藻糖基转移酶Ⅶ(FucT Ⅶ)表达对人结肠癌细胞HT-29与人脐静脉内皮细胞(HUVEC)粘附能力的影响及其机制.本课题构建3对针对FucT Ⅶ基因的RNAi表达载体,并将其转染入人结肠癌细胞HT-29,Western印迹检测FucT Ⅶ及其下游产物sLeX蛋白的变化;实时PCR检测FucT Ⅶ mRNA表达的变化;玫瑰红染色法检测RNAi对HT-29与HUVECs细胞粘附能力的影响.结果显示,3对FucT Ⅶ siRNA表达载体均可有效抑制HT-29细胞FucT Ⅶ mRNA和蛋白表达,以pSilencer 2.0-FucT Ⅶ 2最为有效;与空白细胞组比较,转染pSilencer 2.0-FucT Ⅶ的HT-29细胞表面sLeX表达水平明显下降,以pSilencer2.0-FucT Ⅶ 2最为显著;RNA干扰FucT Ⅶ表达后HT-29细胞和HUVEC之间的粘附能力明显受到抑制.研究表明,RNAi靶向沉默HT-29细胞中FucT Ⅶ基因表达可显著降低其下游产物sLeX的合成,进而抑制HT-29细胞与HUVECs的粘附能力.

In this study,the effect of FucT Ⅶ expression inhibition with RNA interference（RNAi） on the adhesion of human colon carcinoma HT-29 cells to HUVECs was investigated.Three pairs of siRNA were cloned into pSilencer 2.0 vectors and transfected into human colon carcinoma HT-29 cells.The FucT Ⅶ mRNA levels were tested by real-time PCR.The adhesion of transfected HT-29 cells to HUVECs was assayed by the Rose-Bengal method.The sLeX protein levels,downstream of FucT Ⅶ,were assayed by Western blot.The results shown that three siRNA pairs effectively inhibited the FucT Ⅶ and the sLeX expression,especially with the pSilencer 2.0-FucT Ⅶ 2.The RNAi-mediated down-regulation of FucT Ⅶ significantly inhibited the ability of HT-29 cells to adhere to HUVECs.