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目的设计合成对人乙酰肝素酶(heparanase,HPA)基因有特异性抑制作用的短发夹状RNA表达载体,筛选出对HPA沉默效果最佳的质粒。方法根据GenBank数据库提供的人HPA基因核苷酸序列,按短发夹RNA设计原则设计4对特异性寡核苷酸序列及阴性对照,分别克隆到pYr-1.1质粒载体中。用脂质体转染入Hela细胞,分别采用RT-PCR与细胞免疫荧光分析其对HPA在mRNA和蛋白质水平上表达的影响。结果经测序鉴定证实重组质粒构建成功;在构建的4个载体中,HPA-592组Hela细胞和Caski细胞HPA基因mRNA和蛋白质表达水平明显下降,对照组未发生明显改变。结论成功筛选出靶向人HPA的shRNA表达载体,转染宫颈癌Hela细胞和Caski细胞后可高效抑制HPA基因表达。
OBJECTIVE: To design a short hairpin RNA expression vector with specific inhibitory effect on human heparanase (HPA) gene, and to screen out the best plasmids for silencing HPA. Methods According to the nucleotide sequence of human HPA gene provided by GenBank database, 4 pairs of specific oligonucleotide sequences and negative control were designed according to the design principle of short hairpin RNA and cloned into pYr-1.1 plasmid vector respectively. Lipofectamine was transfected into Hela cells. The effects of HPA on the expression of HPA mRNA and protein were analyzed by RT-PCR and immunofluorescence. Results The sequencing results confirmed that the recombinant plasmids were successfully constructed. The expression of HPA mRNA and protein in Hela cells and Caski cells of HPA-592 group was significantly decreased in the four vectors, but not in the control group. Conclusion The shRNA expression vector targeting human HPA was successfully screened. After transfected into cervical cancer Hela cells and Caski cells, HPA gene expression was inhibited efficiently.