Effect of shRNA Inhibiting HIF1α Gene on TIMP1 Expression in RPE Cells
- Author:
Yang CHENG
1
;
Shuiqing ZENG
;
Mingliang LV
Author Information
1. 华中科技大学同济医学院附属医院
- Keywords:
small hairpin RNA;
hypoxic inducible factor 1α;
matrix metalloproteinase tissue inhibitor 1;
hypoxia;
pigmentary epithelia
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2006;26(1):133-136
- CountryChina
- Language:Chinese
-
Abstract:
Small hairpin RNA (shRNA) was used to silence the HIF1α gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2 to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1α mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1α was interfered in RPE cultured under hypoxia (induced by 150 μmol/L CoCl2 ). RT-PCR was employed to detect the expression of HIF1α and TIMP1. The expression levels of HIF1α and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1α mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1α gene silencing in RPE was 83. 4 %. Western blotting revealed that the expression levels of HIF1α protein was dramatically dropped. In addition, RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9 %, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1α mRNA could effectively silence the HIF1α gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
