HIF干扰表达载体psilencer3.0-HIF-α siRNA的构建和鉴定

Construction and identification of psilencer3.0-HIF-α siRNA

背景与目的:肾透明细胞癌是最常见的肾实质恶性肿瘤,生物学行为极为复杂,对放疗和化疗均不敏感。研究发现缺氧诱导因子HIF-1α和HIF-2α与肾透明细胞癌发生发展过程存在相关性。本研究拟通过RNA干扰的方法构建psilencer3.0-HIF-αsiRNA重组质粒,从而为进一步探讨HIF在肾细胞癌发生发展中的功能提供有效的工具。方法:设计并化学合成编码HIF-1α和HIF-2αsiRNA的DNA片段,通过基因重组的方法将其构建进siRNA的表达载体。采用实时定量PCR和Westernblot检测构建成功的siRNA表达载体在mRNA及蛋白水平对目标基因表达的抑制。结果:786-0细胞转染psilencer3.0-HIF-1αsiRNA和psilencer3.0-HIF-2α后,HIF-1α和HIF-2α的mRNA表达的抑制率分别达到了82.1%和87.4%;OS-RC-2细胞转染psilencer3.0-HIF-1αsiRNA和psilencer3.0-HIF-2α后,抑制率分别达到了91.2%和81.2%。在786-0细胞和OS-RC-2细胞中转染HIF-1α干扰质粒和HIF-2α干扰质粒的实验组蛋白表达量较空白对照均有不同程度的减低。结论:构建成功的siRNA表达载体可以有效抑制目标基因HIF-1和HIF-2在mRNA及蛋白水平的表达。

Background and purpose： Carcinoma of the kidney is the most common malignanl renal parenchymal carcinoma： its biological behavior is extremely complicated and is not sensitive to radiotherapy and chemotherapy. Sludies have shown that the hypoxia-inducible factor HIFI-α and H1F2-α are related to the occurrence and development process of the clear-cell carcinonm of kidney. So, we intend to eonstrucl psilencer3.0-HIF-α siRNA recombinant plasmid in this study through RNA inlerference method, thereby providing an effective tnol for further exploration of the role of HIF in the occurrence and development of clear-cell carcinoma of kidney. Methods： Design and chemically synthesize the DNA fragments of coding HIF-lα and HIF-2α siRNA and make them up into siRNA expression vector through gene recombination. Adopting the real-time quantitative PCR and Western blot tested the inhibitive effect of the conslnteted siRNA expression vector on mRNA and protein levels on the target gene expresdon. Results： After 786-0 cells transfecting with psilencer3. O-HIF- lαsiRNA and psilencer3.0-HIF-2α, the inhibitory rate of mRNA expression of HIF-1α and HIF-2α reached 82.1% and 87.4α respectively, and OS-RC-2 cells transfecting psilencer3.0-HIF-1α siRNA and psilencer3. 0-HIF-2α, the inhibition rate reached 91.2% aml 81.2% respectively. The protein expression of the experimental group with 786-0 cells and OS- RC-2 cells transfecting HIF-1α inleclerence plasmid anti HIF-2α interference plasmid were lower at difl＇erent levels than that front the blank control group. Conclusions： The constructed siRNA expression veetor can effectively inhibit the expression of target gene HIF-1 and HIF-2 at mRNA and protein levels.