RNA干扰NF-κBp65抑制膀胱癌BLS细胞的增殖和侵袭  

The inhibition of proliferation and invasion of bladder cancer BLS cells induced by RNA interference on NF-κBp65

作　　者：张丽瑞[1] 陈葳 李琛[1] 李旭 

机构地区：[1]西安交通大学医学院第一附属医院妇产科 [2]医学转化中心,陕西西安710061

出　　处：《肿瘤》2013年第9期758-764,共7页Tumor

基　　金：国家自然科学基金资助项目(编号:81273465,30801446)

摘　　要：目的:探讨RNA干扰核因子-κB(nuclear factor-kappa B,NF-κB)p65对膀胱移行细胞癌BLS细胞增殖和侵袭能力的影响,以及NF-κBp65在膀胱癌组织中的表达及意义。方法:构建靶向NF-κBp65的短发夹RNA(short hairpin RNA,shRNA)重组表达载体pRNAT-U6.1-NF-κBp65-shRNA/Neo,并将其转染至膀胱癌BLS细胞中,蛋白质印迹法验证NF-κBp65的沉默效果。MTT法、平板克隆形成实验及Transwell实验观察pRNAT-U6.1-NF-κBp65-shRNA/Neo转染后BLS细胞的增殖和侵袭能力,蛋白质印迹法检测NF-κB下游分子c-myc、cyclin D1和基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)的表达。免疫组织化学法检测60例膀胱移行细胞癌组织中NF-κBp65和MMP9的表达。结果:成功构建重组载体pRNAT-U6.1-NF-κBp65-shRNA/Neo,并获得稳定转染的NF-κBp65shRNA-BLS细胞。与转染空载体的阴性对照(negative control,NC)NF-κBp65shRNA-NC-BLS细胞比较,NF-κBp65shRNA-BLS细胞的增殖、迁移和侵袭能力下降(P<0.05),同时c-myc、cyclin D1和MMP9蛋白的表达水平下调(P<0.05)。浸润性(T_2~T_4)膀胱癌组织和有淋巴转移的膀胱癌组织中NF-κBp65的表达强度及MMP9的阳性表达率高于浅表性膀胱癌组织(Tis^T_1)和无淋巴转移的膀胱癌组织(P<0.05),且NF-κBp65与MMP9蛋白表达呈正相关(r=0.329,P<0.05)。结论:NF-κB信号通路可能在膀胱癌的增殖和侵袭中发挥作用。Objective： To investigate the effects of RNA interference on NF-κB （nuclear factor-kappa B） p65 on the proliferation and invasion of bladder transitional cell carcinoma BLS cells, and to examine the expression of NF-κBp65 in human bladder transitional cell carcinoma tissues. Methods： The recombinant vector pRNAT-U6.1-NF-κBp65-shRNA/Neo in which the shRNA （short hairpin RNA） targeting NF-κBp65 was established, and then it was transfected into the BLS cells. The efficacy of gene silencing was confirmed by Western blotting. The proliferation, migration and invasion abilities of BLS cells after transfection with pRNAT-U6.1-NF-κBp65-shRNA/Neo were detected by MTT assay and colony-forming assay, respectively. The downstream targets of NF-κB, including c-myc, cyclin D1 and MMP9 （matrix metalloproteinase 9） were examined by Western blotting. The expressions of NF-κBp65 and MMP9 proteins in 60 cases of bladder transitional cell carcinoma tissues were detected by immunohistochemistry. Results： The recombinant vector pRNAT-U6.1-NF-κBp65-shRNA/Neo was successfully constructed, and the stably transfected cells were established. As compared with NC （negative control） NF-κBp65shRNA-NC-BLS cells which were tranfected with an empty vector, the abilities of proliferation, colony formation, migration and invasion of the NF-κBp65shRNA-BLS cells were obviously decreased （P〈0.05）, as well as the expression levels of c-myc, cyclin D1 and MMP9 proteins were down-regulated （P〈0.05）. The expression levels of NF-κBp65 and MMP9 in stages T2-4 bladder transitional cell carcinoma and the cancer with lymphatic metastasis were higher than those in stages Tis-T~ bladder transitional cell carcinoma and the cancer without lymphatic metastasis （P 〈 0.05）. There was an obviously positive correlation between the NF-κBp65 and MMP9 expressions （r = 0.329, P 〈 0.05）. Conclusion： NF-κB signal transduction way may be related to the proliferation and invasion of bladder cancer cells.

关 键 词：膀胱肿瘤 核转录因子ΚB 细胞增殖 肿瘤浸润 

分 类 号：R737.14[医药卫生—肿瘤]