乙酰肝素酶基因siRNA表达载体的构建及其沉寂作用

目的:构建针对人乙酰肝素酶(HPSE)基因的小干扰RNA(siRNA)及其表达载体,转染细胞后观察其对HPSE基因的干扰作用。方法:设计HPSE靶向的发夹状siRNA,合成两条互补的寡核苷酸链,退火后连接入pRNATU6.1载体,转化扩增后进行序列测定。用脂质体包裹转染人恶性黑素瘤细胞A375,采用半定量PCR测定HPSE基因RNA水平变化,Western blot检测HPSE蛋白表达的变化。结果:将针对HPSE基因的siRNA的双链寡核苷酸片段克隆到pRNATU6.1载体,经过阳性菌落PCR鉴定与测序,结果正确;转染A375细胞后,半定量PCR和Western blot检测显示,HPSE基因和蛋白的表达水平明显降低(P<0.05)。结论:成功构建了针对HPSE基因的siRNA载体,转染细胞后可抑制HPSE的表达。

Construction of heparanase gene-targeted small interfering RNA and its gene silencing effect

Objective: To construct heparanase gene-targeted small interfering RNA(siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant melanoma A375 cell.Methods: Heparanase gene-targeted hairpin siRNA was designed,two complementary oligonucleotide strand was synthesized and inserted into pRNATU6.1 vector,which was then identified by PCR and sequencing.Human malignant melanoma A375 cells were transfected with the constructed vector using lipofectamine method.Semi-quantitative PCR was performed to evaluate the heparanase-mRNA expression levels,and Western blot was performed to evaluate the expression of heparanase protein.Results: The vector containing siRNA was identified by PCR and sequencing;the results of semi-quantitative PCR and Western blot showed that the expression levels of both heparanase RNA and protein in transfected A375 cells were decreased significantly(P<0.05). Conclusion: The heparanase gene-targeted siRNA and its vector were successfully constructed,which can reduce the heparanase gene and protein expression in transfected cells.