Neuroscience Bulletin 2009;25(3):122-130

The construction of siRNA plasmid targeting mouse HIF-1alpha and in vitro study of its inhibition effect.

Zhen-Yu DING 1 ; Ze-Gui LI ; Yi-Zhan XING ; Hua JI ; Hong-Li LI ; Zhi-Jie CHANG

Affiliations

+expand

Country

China

Language

English

MeSH

Abstract

OBJECTIVETo construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.

METHODSThree RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated.

RESULTSAll the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way.

CONCLUSIONPlasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.