目的:研究siRNA干扰葡萄糖-6-磷酸脱氢酶(G6PD)基因表达的LoVo细胞在低氧环境中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平的变化及其对低氧诱导因子1α(HIF-1α)表达的影响.方法:构建靶向G6PD基因的siRNA干扰质粒载体,并转染人结肠癌细胞株(LoVo),用限制性内切酶法结合DNA测序鉴定特异重组子,RT-PCR检测G6PD mRNA表达,结合G6PD活性测定判断其干扰效率;以未转染LoVo细胞为对照,进一步观察G6PD沉默的瘤细胞在低氧环境下NADPH水平的变化,RT-PCR检测HIF-1α mRNA表达水平,Western blotting检测HIF-1α蛋白水平.结果:成功构建靶向G6PD的siRNA重组质粒载体并稳定转染LoVo细胞.与未转染组细胞相比,RNA干扰G6PD使mRNA表达下降43%,酶活性下降63.5%,细胞内NADPH水平显著下降(41% vs 100%,P<0.05),但HIF-1α mRNA表达水平无明显变化,而HIF-1α蛋白显著降低.结论:低氧环境下,G6PD基因可能通过下调NADPH水平从而降低HIF-1α的稳定性,导致其水平下降,进而影响肿瘤细胞的低氧反应.%AIM: To study the effects of glucose - 6 - phosphate dehydrogenase ( G6PD ) silencing by small interference RNA( siRNA ) on the levels of reduced nicotinamide - adenine dinucleotide phosphate ( NADPH ) and hypoxia - inducible factor 1α( HIF - 1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed.The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells.The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD.LoVo cells were cultured in vitro under hypoxic condition.NADPH levels were determined.The mRNA and protein levels of HIF - 1α were analyzed by RT - PCR and Western blotting, respectively.RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells.Compared with untransfected cells, the mRNA expression of G6PD in transfected cells was decreased by 43％ and G6PD activity was decreased by 63.5％.Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased ( 41％ vs 100％ , P ＜0.05 ).HIF - 1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells.CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF - 1α stability in cancer cells under hypoxic condition.By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.
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