HIF-1α特异性RNA干扰表达载体的构建及体外效应研究

The construction and identification of siRNA expression vector against hypoxia inducible factor-1α in vitro

目的己知缺氧诱导因子-1α(HIF-1α)在缺氧状态下可通过调节内皮素-1(ET-1)的升高而参与肺血管内皮细胞损伤及肺动脉高压,最终导致肺出血。RNA干扰(RNAi)是双链RNA介导的序列特异性转录后同源靶基因沉默效应,目前RNAi技术已成为一种研究基因功能的有力工具,故拟通过构建人HIF-1α基因的特异性小干扰RNA(siRNA)真核表达载体,观测该载体在缺氧状态下对HIF-1α基因的干扰作用及对ET-1的抑制作用。方法选择6个(a^f)可能的人HIF-1α siRNA干扰位点,设计合成相应的特异性互补寡聚核苷酸链(A^F),采用基因克隆技术,将合成的(A^F)链序列定向插入真核表达载体中,构建成重组质粒HIF-1αsiRNA真核表达载体(A′~F′),通过脂质体法转染至体外人脐静脉内皮细胞(HUVECs)48h,再以CoCl2(100μM)模拟缺氧刺激3h后,观测siRNA对HIF-1αmRNA和HIF-1α蛋白表达的抑制效应及ET-1水平。结果①成功构建了分别针对6个HIF-1αsiRNA干扰位点(a^f)的重组质粒HIF-1αsiRNA真核表达载体(A′~F′);②该6组真核表达载体转染HUVECs并经CoCl2模拟缺氧刺激后,与未转染组比较,结果显示B′及D′组明显抑制HIF-1α mRNA及HIF-1α蛋白的表达,并部分抑制ET-1表达,而其余4组与未转染组比较并无差异。结论针对b及d干扰位点的B′及D′特异性siRNA真核表达载体,能明显干扰HIF-1α基因的表达,进而抑制ET-1的释放。该实验为下一步用B′、D′特异性siRNA真核表达载体在动物体内研究HIF-1α与新生儿缺氧性肺出血关系奠定基础。

Objective Previous studies have suggested that under hypoxic conditions hypoxia inducible factor-1α （HIF-1α） 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 pest-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-1α gene （pSIREN-Shuttle HIF-1α siRNA）in order to observe its silencing effect on HIF-1α under hypoxic conditions. Methods Six potential siRNA target sites （a-f）specific to human HIF-1α 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 oligonueleotide into RNAi-Ready pSIREN vector. The recombinant vectors were then transfeeted into the cultured human umbilical vein endothelial cells （HUVECs）. After 48 hrs of culture, the cells were treated with CoCl2 （100 μM） for 3 hrs. Expression of HIF-1α 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-1α siRNA eukaryotic expression vectors A＇-F＇ respectively aiming at sites （a-f） were constructed successfully. Compared to the non-transfection group, lipesome- mediated gene transfection of pSIREN-Shuttle HIF-1α siRNA expression vectors into HUVECs obviously down-regulated the mRNA and protein levels of HIF-1α, and partly decreased the ET-1 level in the B＇and D＇ transfection groups. Conclusions The specific pSIREN-Shuttle HIF-1α siRNA expression vectors B＇ and D＇ aiming at b and d sites can inhibit the expression of HIF-1α, thus decreasing the level of its target gene ET-1. This may he helpful to study the relationship between HIF-1α and neonatal pulmonary hemorrhage in vivo in future.