The construction of siRNA plasmid targeting mouse HIF-1alpha and in vitro study of its inhibition effect.
- Author:
Zhen-Yu DING
1
;
Ze-Gui LI
;
Yi-Zhan XING
;
Hua JI
;
Hong-Li LI
;
Zhi-Jie CHANG
Author Information
- Publication Type:Journal Article
- MeSH: Analysis of Variance; Animals; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression; drug effects; Gene Silencing; physiology; Green Fluorescent Proteins; genetics; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; genetics; metabolism; Mice; Plasmids; genetics; RNA, Small Interfering; genetics; pharmacology; Transfection; methods
- From:Neuroscience Bulletin 2009;25(3):122-130
- CountryChina
- Language:English
-
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.
