Title: The construction of siRNA plasmid targeting mouse HIF-1α and in vitro study of its inhibition effect
Abstract: To construct effective RNA-interference plasmids targeting mouse HIF-1α gene and testify their effects and specificities in interfering HIF-1α expression. Three RNA-interference plasmids targeting mouse HIF-1α gene, pBS/U6/HIF-1α-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-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated. All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-II, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-II could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dose-dependent way. Plasmid pBS/U6/HIF1αi-II constructed in our study can effectively and specifically inhibit HIF-1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1αi-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.