Hypoxia inducible factor-1α gene silencing aggravates growth inhibition and necrosis of human proximal renal tubular epithelial cell under hypoxia
10.3760/cma.j.issn.1001-7097.2010.07.010
- VernacularTitle:小分子RNA干扰沉默缺氧诱导因子1α加重缺氧状态肾小管上皮细胞的生长抑制和坏死
- Author:
Yue CHEN
;
Suhua JIANG
;
Jiaming ZHU
;
Yihong ZHONG
;
Chensheng FU
;
Hong LIU
;
Yi FANG
;
Xiaoqiang DING
- Publication Type:Journal Article
- Keywords:
Hypoxia-inducible factor 1,alpha subunit;
RNA interference;
Anoxia;
Renal tubular epithelial cells
- From:
Chinese Journal of Nephrology
2010;26(7):530-536
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effect of hypoxia inducible factor-1α silencing by siRNA on proliferation, apoptosis and necrosis of human renal proximal epithelial cell ( HK-2 )under hypoxia. Methods CoCl2 was used to mimic hypoxia, and siRNA technique was used to silence HIF-1α. HK-2 cells were divided into five groups, including normal culture group,hypoxia culture group, transfection reagent group, negative control group and HIF-1α siRNA group.MTT assay was used to detect the growth inhibition ratio of cells. TUNEL and caspase-3 quantity was used to detect apoptosis. LDH activity in supernatant was detected to evaluate cell necrosis.Real-time PCR and Western blotting were used to detect mRNA and protein of HIF-1α, HIF-2α,glucose transporter 1(Glut-1) and vascular endothelial growth factor (VEGF). Results (1) The transfection efficiency of siRNA was 95%-100%. Under normoxia, the efficiency of siRNA silencing HIF-1α gene reached to 70%, while under hypoxia, it was 97%. (2) The growth inhibition ratio of cells in HIF-1α siRNA group was significantly higher than that of other groups including hypoxia culture group, transfection reagent group and negative control group (all P＜0.05). No significant difference was found in apoptotic ratio of the other four groups except normal culture group (P＞0.05). The LDH level in HIF-1α siRNA group was significantly higher than that of hypoxia culture group, transfection reagent group and negative control group (P＜0.05). (3) The expression of HIF1α, Glut-1, VEGF mRNA and protein in HIF-1α siRNA group was significantly lower than that of hypoxia culture group, transfection reagent group and negative control group (P＜0.05). No significant difference was observed on the level of HIF-2α mRNA and protein in the other four groups except normal culture group (P＞0.05). Conclusion HIF-1α gene silencing can inhibit the mRNA and protein expression of Glut-1 and VEGF, and can aggravate growth inhibition and necrosis of HK-2 cells under hypoxia.
