[The construction and identification of siRNA expression vector against hypoxia inducible factor -1 alpha in vitro].
OBJECTIVE
Previous studies have suggested that under hypoxic conditions hypoxia inducible factor-1 alpha (HIF-1 alpha) contributes to the progression of neonatal pulmonary hemorrhage (NPH) by increasing the expression of endothelin-1 (ET-1) gene. RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene-silencing mediated by double-stranded RNA. This new gene-silencing technique has recently been shown to be a powerful approach for studying gene function. The aim of this study was to construct the small interfering RNA (siRNA) eukaryotic expression vectors specific to human HIF-1 alpha gene (pSIREN-Shuttle HIF-1 alpha siRNAin order to observe its silencing effect on HIF-1 alpha under hypoxic conditions.


METHODS
Six potential siRNA target sites (a-fspecific to human HIF-1 alpha gene were designed and synthesized to two complementary oligonucleotides (A-F) for each siRNA target site. Using a gene recombination technique, the recombinant expression vectors (A-F') were constructed by cloning the double strands oligonucleotide into RNAi-Ready pSIREN vector. The recombinant vectors were then transfected into the cultured human umbilical vein endothelial cells (HUVECs). After 48 hrs of culture, the cells were treated with CoCl2 (100 mu M) for 3 hrs. Expression of HIF-1 alpha mRNA and protein was detected using RT-PCR and Western blot. ET-1 level in cell culture supernates was detected using ELISA.


RESULTS
The recombinant HIF-1 alpha siRNA eukaryotic expression vectors A'-F'respectively aiming at sites (a-f) were constructed successfully. Compared to the non-transfection group, liposome-mediated gene transfection of pSIREN-Shuttle HIF-1 alpha siRNA expression vectors into HUVECs obviously down-regulated the mRNA and protein levels of HIF-1 alpha, and partly decreased the ET-1 level in the B' and D' transfection groups.


CONCLUSIONS
The specific pSIREN-Shuttle HIF-1 alpha siRNA expression vectors B' and D' aiming at b and d sites can inhibit the expression of HIF-1 alpha, thus decreasing the level of its target gene ET-1. This may be helpful to study the relationship between HIF-1 alpha and neonatal pulmonary hemorrhage in vivo in future.
He
Liu-Fang
LF
Department of Neonatal Intensive Care Unit, Guangzhou Children's Hospital, Guangzhou 510120, China.
Chen
Ke-Zheng
KZ
D001483:Base Sequence; D019332:Endothelin-1 / Q000032:analysis; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000235:genetics; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics*
D019332:Endothelin-1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering