Smad4基因敲低对结肠癌阿霉素化疗敏感性的影响  

Smad4 gene knockdown enhances chemosensitivity of colon cancer cells to doxorubicin

作　　者：李金鹏[1] 刘昊[1] 于皆平[1] 于红刚[1] 

机构地区：[1]武汉大学人民医院消化内科,湖北省武汉市430060

出　　处：《世界华人消化杂志》2014年第19期2746-2751,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.81272693~~

摘　　要：目的:探讨应用小发卡RNA(small hairpin RNA,shRNA)干扰技术敲低Smad4基因对结肠癌细胞阿霉素(doxorubicin)化疗敏感性的影响.方法:应用shRNA干扰技术敲低人结肠癌H C T116细胞Smad4基因,分空白细胞对照Control组,shRNA阴性对照RNAi-NC组和shRNA干扰RNAi-Smad4组,采用终浓度为50 nmoL/L doxorubicin用7 d,蛋白免疫印迹和逆转录聚合酶链反应鉴定Smad4敲低效果.MTT检测细胞存活率.酶联免疫吸附测定检测转化生长因子β(transforming growth factor beta 1,TGF-β1)的表达水平.蛋白免疫印迹检测多药耐药基因糖蛋白(multi drugs resistance gene plasma membrane glycoprotein,MDR P-g p),上皮细胞间质细胞样转化相关的标志物E-cadherin.Vimentin和相关的转录因子Snail、Slug、Smad2/3和磷酸化Smad2/3的蛋白表达.结果:Smad4 shRNA能够显著敲低Smad4基因的表达.MTT结果显RNAi-Smad4组较RNAi-N C组的细胞存活率降低,且呈时间依赖性.50 nmoL/L阿霉素作用于癌细胞7 d后,ELISA结果提示阿霉素能显著提高RNAi-Smad4组和RNAi-NC组的TGF-β1的表达(P<0.05).蛋白免疫印迹提示MDR p-gp、Vimentin、Snail和Slug的表达RNAi-NC组较对照组升高而RNAi-Smad4能抑制这种高表达,E-cadherin蛋白RNAi-NC组较对照组减低而RNAi-Smad组则能升高该蛋白的表达;阿霉素处理后不影响Smad2/3的表达,但提高了RNAi-Smad4组和RNAi-NC组的磷酸化Smad2/3的表达.结论:敲低Smad4基因以阻断TGF-β/Smad4信号通路可提高结肠癌的阿霉素化疗敏感性.AIM： To evaluate whether small hairpin RNA（shRNA）-mediated knockdown of the Smad4 gene influences resistance of colon cancer cells to doxorubicin in vitro.METHODS： An Smad4 shRNA was stably trans-fected into human colon cancer HCT116 cells to silence the Smad4 gene. Cells were basically divided into three groups： shRNA-negative control cells（RNAi-NC）, shRNA-Smad4 cells（RNAi-Smad4） and HCT116 cells untreated by doxorubicin（CONTROL）. Doxorubicin（50 nmoL/L） was applied to treat cells for 7 d. The expression of Smad4 was examined by Western blot and RT-PCR to test shRNA transfection ef-ficiency. Cell viability was determined by MTT assay, the concentration of transforming growthfactor beta 1（TGF-β1） was measured by ELISA, and expression of multidrug resistance gene plasma membrane glycoprotein（MDR P-gp）, epithelial mesenchymal transition（EMT） related markers E-cadherin and vimentin, related tran-scription factors Snail and Slug, Smad2/3 and phosphorylation of Smad2/3 expression were detected by Western blot.RESULTS： The protein and mRNA levels of Smad4 were significantly reduced after Smad4 shRNA transfection. After doxorubicin（50 nmoL/L） administration for 7 d, MTT assay showed that cell viability ratio in the RNAi-Smad4 group was lower than that in the RNAi-NC group, and declined in a time dependent manner. ELISA assay revealed that TGF-β1 concentration in the RNAi-Smad4 and RNAi-NC groups were significantly augmented by doxorubicin（P〈0.05）. Western blot results indicated that protein expression of MDRP-gp, vimentin, Snail and Slug in the RNAi-NC group was higher than that in the CONTROL group, but RNAi-Smad4 inhibited such increases. In contrast, expression of E-cadherin in the RNAi-NC group was lower than that in the CONTROL group, but RNAi-Smad4 enhanced its expres-sion. Doxorubicin administration did not change the expression of Smad2/3 but enhanced phos-phorylation of Smad2/3 expression in the RNAi-Smad4 and RNAi-NC groups.CONCLUSION： Smad4 gene knockdown t

关 键 词：SMAD4 RNA干扰 阿霉素 结肠癌 耐药性 

分 类 号：R735.35[医药卫生—肿瘤]