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本研究根据pSUPER.retro.puro载体的特有结构,构建缺氧诱导因子1α(HIF-1α)的RNA干扰(RNAi)表达载体,将其与GFP共转染前列腺癌PC-3细胞36 h后转染效率为(87.15±2.36)%;通过与对照组的比较,质粒转染PC-3细胞48 h后可显著降低胞内HIF-1αmRNA及蛋白的表达;利用重组质粒的嘌呤霉素的抗药性,加药筛选2周,可观察到单克隆生成;收集稳定转染得到的pSUPER-siHIF-1α/PC-3细胞株,再次通过West blot检测到PC-3细胞的HIF-1α蛋白表达亦显著降低。结果表明,利用RNAi技术可成功构建抑制前列腺癌HIF-1α表达的si RNA重组表达载体,为研究HIF-1α在前列腺癌发病机理及增殖、转移中的功能奠定了基础,同时可以用于其在其他肿瘤的功能研究。
In this study, according to the unique structure of pSUPER.retro.puro vector, RNA interference (RNAi) expression vector of hypoxia inducible factor 1α (HIF-1α) was constructed and transfected into PC-3 cells with GFP for 36 h (87.15 ± 2.36)%. Compared with the control group, the expression of HIF-1αmRNA and protein in PC-3 cells transfected into PC-3 cells can be significantly reduced by 48 h. The puromycin resistance of the recombinant plasmids , And the monoclonal antibody was observed after 2 weeks of drug addition. The pSUPER-siHIF-1α / PC-3 cell line was collected stably and the expression of HIF-1α in PC-3 cells was also detected by Western blot reduce. The results showed that RNAi technology can successfully construct a si RNA recombinant expression vector that inhibits the expression of HIF-1α in prostate cancer, which lays the foundation for studying the function of HIF-1α in the pathogenesis, proliferation and metastasis of prostate cancer. At the same time, Other tumor function studies.