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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 effect of nuclear factor-kappa B (NF-κB) p65 on the proliferation and invasiveness of bladder transitional cell carcinoma (BLS) cells and the expression and significance of NF-κBp65 in bladder cancer. Methods: The shRNA expression vector pRNAT-U6.1-NF-κBp65-shRNA / Neo targeting NF-κBp65 was constructed and transfected into BLS cells of bladder cancer. The protein The silencing effect of NF-κBp65 was verified by western blotting. The proliferation and invasion ability of BLS cells after transfection with pRNAT-U6.1-NF-κBp65-shRNA / Neo were observed by MTT assay, plate clone formation assay and Transwell assay. The expressions of c-myc, cyclin D1 And matrix metalloproteinase 9 (MMP9). Immunohistochemistry was used to detect the expression of NF-κBp65 and MMP9 in 60 cases of bladder transitional cell carcinoma. Results: The recombinant vector pRNAT-U6.1-NF-κBp65-shRNA / Neo was successfully constructed and stably transfected NF-κBp65shRNA-BLS cells were obtained. Compared with the negative control (NC) NF-κBp65shRNA-NC-BLS cells transfected with empty vector, the proliferation, migration and invasion of NF-κBp65shRNA-BLS cells decreased (P <0.05) The expressions of D1 and MMP9 were down-regulated (P <0.05). The positive expression rate of NF-κBp65 and the positive rate of MMP9 in invasive T_2 ~ T_4 bladder cancer and lymph node metastasis were higher than those in superficial bladder cancer (Tis ~ T_1) and without lymph node metastasis (P <0.05), and there was a positive correlation between NF-κBp65 and MMP9 (r = 0.329, P <0.05). Conclusion: NF-κB signaling pathway may play a role in the proliferation and invasion of bladder cancer.