HIF-1α的RNA干涉可引起缺氧条件下人角膜上皮细胞VEGF表达下调和PEDF表达上调

Down-regulation of VEGF expression and up-regulation of PEDF expression in human corneal epithelial cells under hypoxic condition by RNA interference of HIF-1α

目的探讨shRNA干扰HIF-1α表达对缺氧条件下人角膜上皮细胞(h CEC)血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)表达的影响。方法针对HIF-1αmRNA序列靶点设计及合成3个小发夹RNA(shRNA),分别在体外转染自发永生化人角膜上皮细胞(S-ihCEC),转染后的S-ihCEC在1 mmol/L Co Cl2中缺氧诱导培养6 h,实时定量PCR检测HIF-1α、VEGF、PEDF的mRNA表达,Western blot法检测HIF-1α、VEGF的蛋白表达,ELISA检测PEDF的水平。结果与缺氧组比较,转染HIF-1αshRNA的S-ihCEC中HIF-1αmRNA表达下调、HIF-1α蛋白表达下调,VEGF mRNA表达下调、VEGF蛋白表达下调,PEDF mRNA表达上调(1. 22±0. 18)倍,PEDF水平升高。结论针对HIF-1α的shRNA能有效干扰S-ihCEC中HIF-1α的表达,并下调VEGF表达、上调PEDF的表达。

Objective To investigate the effect of short hairpin RNA( shRNA) targeting hypoxia-inducible factor-1α( HIF-1α) on the expression of vascular endothelial growth factor( VEGF) and pigment epithelium derived factor( PEDF) inspontaneously immortalized human corneal epithelial cells( S-ihCECs) under hypoxic condition. Methods Three kinds ofshRNAs targeting the sites of HIF-1α mRNA were designed and synthesized,and then transfected into S-ihCECs in vitro.After transfection,the cells were exposed to 1 mmol/L Co Cl2 for 6 hours. HIF-1α,VEGF and PEDF mRNA expressions weredetected by real-time quantitative PCR. HIF-1α and VEGF protein levels were analyzed by Western blotting. PEDF level wastested by ELISA. Results After the shRNA interference,the expressions of HIF-1α and VEGF mRNA decreased by( 70. 9 ±2. 8) % and( 51. 9 ± 15. 7) %,respectively when compared with the hypoxia group. The expressions of HIF-1α and VEGFproteins were both down-regulated. The expression of PEDF mRNA increased( 1. 22 ± 0. 18) fold and the protein wasup-regulated. Conclusion ShRNA targeting HIF-1α can effectively silence the expression of HIF-1α,reduce the expressionof VEGF and up-regulate the expression of PEDF.