缺氧诱导因子1α的RNAi表达载体构建及其在前列腺癌PC-3细胞研究中的作用

Construction of Expression Vector for RNA Interference of Hypoxia-inducible Factor 1α and Its Function in the Study of Prostatic Carcinoma Cell Line PC-3

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。

According to the specific construction of the pSUPER.retro.puro vector,RNA interfering with the hypoxia-inducible factor 1α（HIF-1α） plasmid was constructed in this research programme.The efficiency was（87.15±2.36）% and green fluorescent protein was observed after 36 hours when the constructed plasmid co-transformed into the prostatic carcinoma cell line PC-3;compared with the control groups,this constructed plasmid could reduce the expression of total RNA and protein in PC-3 cells significantly after 48 hours. The cells were checked out by the plasmid resistance against the puromycin biotin,the clones of which were selected and enlarged for two weeks,then the cell strain of pSUPER-siHIF-1α/PC-3 was collected.The HIF-1α protein expression of the pSUPER-siHIF-1α/PC-3 cells of also decreased significantly.The results showed that the RNA interference could be used in the construction of expression vector of constructed siRNA inhibiting the expression of HIF-1α with PC-3 cells,which is the basis of researching the pathology,multiplication,and metastasis of HIF-1α in the prostatic carcinoma and other cancers.