腺病毒载体介导的RNAi对SK-BR-3细胞c-Met表达的影响

目的:通过腺病毒载体介导的RNA干扰技术抑制乳腺癌细胞HGF受体c-Met的表达,抑制乳腺癌细胞增殖、诱导细胞凋亡。方法:PCR法获得人U6启动子及带有c-Met反向互补靶序列的片段HU6shmet;利用腺病毒载体将其传递至SK-BR-3细胞;RNA狭缝杂交检测SK-BR-3细胞c-Met mRNA水平,Western印迹检测c-Met蛋白水平。结果:获得了带有人U6启动子及c-Met反向互补靶序列的重组腺病毒载体rAdUshmet1和rAdUshmet2。转导了重组腺病毒的SK-BR-3细胞的c-Met mRNA和蛋白相对表达量均有所下降。结论:腺病毒载体rAdUshmet通过抑制HGF受体c-Met表达,能在一定程度上阻断HGF-c-Met信号转导通路,有可能成为对乳腺癌进行基因治疗的有效载体。

Influence of adenovirus vector-mediated RNAi on expression of c-Met in SK-BR-3 cells

Objective: To inhibit the expression of hepatocyte growth factor (HGF) receptor-c-Met by RNAi technique mediated by adenovirus vector, so as to inhibit tumor cell proliferation and induce tumor cell apoptosis. Methods: Human U6 promotor and the fragment including c-Met reverse complement target sequence were obtained by PCR. The fragment was packaged in adenovirus and transduced into SK-BR-3 cells. c-Met mRNA level was assayed by slit hybridization and the protein level was assayed by Western blot. Results: The recombinated adenovirus vector (named rAdUshmet1 and rAdUshmet2), harboring human U6 promotor and c-Met reverse complement target sequence, were obtained. The c-Met mRNA and protein expression levels were reduced in the SK-BR-3 cells transducted with the rAdUshmet1 and rAdUshmet2. Conclusion: The c-Met short hairpin RNA mediated by adenovirus vector can inhibit c-Met expression, block the HGF-c-Met signal pathway partially and may act as an effective vector for gene therapy of breast cancer.