缺氧诱导因子-1α小干扰RNA对HaCaT细胞VEGF表达的影响

LI Yong-fian, ZU Xu-yu, ZHANG Gui-ying, XIAO Rong, WEN Hai-quan. Department of Dermatology, First Affiliated Hospital of Nanhua University, Hengyang 421001, Hunan, China

目的探讨缺氧诱导因子-1α（HIF-1α）RNA干扰对低氧条件下HaCaT细胞HIF-1α和血管内皮细胞生长因子（VEGF）表达的影响。方法将HaCaT细胞分为4组：常氧对照组（无干预因素）、低氧组（缺氧培养24h）、脂质体对照组（转染空载脂质体后缺氧培养24h）、RNA干扰组（转染脂质体介导的RNA干扰序列后缺氧培养24h）。荧光实时定量PCR法检测各组HaCaT细胞HIF-1“和VEGFmRNA表达水平，Western印迹检测各组HaCaT细胞HIF-1α和VEGF蛋白表达水平。结果低氧组和常氧对照组HIF—1αmRNA的表达水平分别为0．907±0．032和0．878±0．034，两者比较差异无统计学意义（F=1．108，P〉0．05），而低氧组VEGFmRNA和HIF-1α及VEGF蛋白的表达水平分别为0．935±0．032和0．813-4-0．047，0．791±0．030，均较常氧对照组（分别为0．652±0．053、0．236±0．014和0.316±0．013）明显增高（P值均〈0．05）；RNA干扰组HIF—1αmRNA及蛋白的表达水平为0．230±0．044和0．213±0．026，VEGF为0．497±0．033和0．249±0．028，均较脂质体对照组（分别为0．978±0．030、0．817±0．049和0．8064-0．040、0．833±0．052）显著降低（P值均〈0．05）。结论低氧可以使HaCaT细胞HIF-1α和VEGF的表达增加，而抑制HIF-1α的表达可以使低氧条件下HaCaT细胞VEGF的表达减少。

Objective To observe the effects of hypoxia inducible factor-1 alpha （HIF-1α）-targeting small interfering RNA （siRNA） on the expression of HIF-1α and vascular endothelial growth factor （VEGF） in HaCaT cells under hypoxic conditions. Methods HaCaT cells were cultured and divided into four groups, normal control group （without any treatment）, hypoxia group （cultured under hypoxic conditions for 24 hours）, liposome control group （transfected with liposome followed by hypoxic culture for 24 hours）, RNA interference group （transfected with HIF-1α-targeting siRNA/liposome complexes followed by hypoxic Culture for 24 hours）. Fluorescence real-time quantitative PCR was utilized to determine HIF-1α and VEGF mRNA expres- sion in HaCaT ceils, and Western blot to detect HIF-1α and VEGF protein expression. Results No significant difference was observed in the mRNA expression of HIF-1α between the hypoxia group and normal control group （0.907 ± 0.032 vs. 0.878 ± 0.034, F =1.108, P 〉 0.05）, while the expression levels of VEGF mRNA, HIF-1α and VEGF proteins were significantly higher in the hypoxia group than in the normal control group （0.935 ± 0.032 vs. 0.652±0.053, 0.813 ± 0.047 vs. 0.236 ±0.014, 0.791 ±0.030 vs. 0.316 ± 0.013, all P 〈 0.05 ）. A significant decline was noted in the mRNA and protein expression levels of VEGF （0.230 ± 0.044 vs. 0.978 ± 0.030, 0.213 ±0.026 vs. 0.817 ＋ 0.049, both P 〈 0.05） and HIF-lct （0.497± 0.033 vs. 0.806± 0.040, 0.249± 0.028 vs. 0.833 ± 0.052, both P 〈 0.05） in the RNA interference group than in the liposome control group. Conclusions Hypoxia may enhance the expression of HIF-1α and VEGF in HaCaT cells, and to inhibit the HIF-1α expression may suppress the expression of VEGF in HaCaT cells under hypoxia.