The construction of siRNA plasmid targeting mouse HIF-1alpha and in vitro study of its inhibition effect.
OBJECTIVE
To construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression.


METHODS
Three 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.


RESULTS
All 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.


CONCLUSIONS
Plasmid 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.
Ding
Zhen-Yu
ZY
Department of pharmacognosy, College of pharmacy, Third Military Medical University, Chongqing 400038, China.
Li
Ze-Gui
ZG
Xing
Yi-Zhan
YZ
Ji
Hua
H
Li
Hong-Li
HL
Chang
Zhi-Jie
ZJ
D000704:Analysis of Variance; D000818:Animals; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins