小RNA干扰沉默CD147基因对膀胱癌T-24细胞生物学行为的影响

Gene silencing of CD147 by adenovirus-mediated small interfering RNA interference influences the biological behaviors of bladder cancer T-24 cells

目的:探讨重组腺病毒介导的小RNA干扰(small interfering RNA,siRNA)沉默CD147基因表达对人膀胱癌T-24细胞增殖、迁移及侵袭能力的影响。方法:构建针对CD147基因的siRNA重组腺病毒表达载体Ad-CD147siRNA,感染人膀胱癌T-24细胞。实验设空白对照组(Mock)、阴性对照组(Ad-siControl)和干扰组(Ad-CD147siRNA)。采用RT-PCR检测CD147和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA的表达水平;蛋白质印迹法检测CD147蛋白的表达水平;MTT法检测细胞增殖的情况,Transwell小室法和划痕实验检测对细胞侵袭和迁移能力的影响;明胶酶谱实验检测基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和MMP-9的分泌情况;ELISA法检测VEGF蛋白分泌水平。结果:成功构建了Ad-CD147siRNA腺病毒表达载体,Ad-CD147siRNA可使T-24细胞CD147mRNA和蛋白表达量下调>90%(P<0.01),细胞增殖速率下降>40%(P<0.01),细胞侵袭力和迁移力明显下降(P<0.01),MMP-2和MMP-9的分泌量分别下降了68.5%和37.1%(P<0.01),VEGF mRNA和蛋白表达水平分别下降了57.2%(P<0.01)和56.5%(P<0.01)。结论:Ad-CD147siRNA能有效抑制T-24细胞中CD147基因的表达,继而降低细胞增殖、迁移及侵袭能力。因此,CD147有可能成为膀胱癌基因治疗的一个新靶点。

Objective：To investigate the effects of gene silencing of CD147 by adenovirus-mediated small interfering RNA（siRNA） interference on the proliferation,migration and invasion of bladder cancer T-24 cells in vitro.Methods：The recombinant adenoviral vector encoding CD147 siRNA was constructed,and then the T-24 cells were infected with this recombinant adenoviral vector.The blank control（Mock）,negative control（Ad-siControl） and siRNA interference（Ad-CD147 siRNA） groups were designed in this study.The expression levels of CD147 and vascular endothelial growth factor（VEGF） mRNAs were detected by RT-PCR,and the expression level of CD147 protein was detected by Western blotting.The cell proliferation was detected by MTT method.The invasion and migration capabilities were evaluated by using Transwell chamber migration assay and scratch wound healing assay,respectively.The secretion levels of matrix metalloproteinase（MMP）-2 and MMP-9 were detected by gelatin zymography.The secretion level of VEGF protein was examined by ELISA.Results：The recombinant adenoviral vector Ad-CD147 siRNA was successfully constructed and infected into T-24 cells.The expression levels of CD147 mRNA and protein in T-24 cells were decreased by more than 90%（P〈0.01）,and the proliferative rate was decreased by more than 40%（P〈0.01）.The invasion and migration capabilities were both decreased significantly（P〈0.01）.The secretion levels of MMP-2 and MMP-9 were reduced by 68.5% and 37.1%,respectively（P〈0.01）.The VEGF mRNA and protein levels were reduced by 57.2%（P〈0.01） and 56.5%（P〈0.01）,respectively.Conclusion：Ad-CD147 siRNA can efficiently inhibit the expressions of CD147 protein and mRNA,which results in decreased capacities of proliferation,migration and invasion of T24 cells.CD147 maybe become a new potential target in the treatment of bladder cancer.