Construction of a lentiviral RNA interference system targeting heparanase based on miR30 and its silencing effect / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.</p><p><b>METHODS</b>Three heparanase-shRNA structures were designed based miR30. The targeting fragments were obtained by PCR, then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA, which was identified by PCR and sequencing. The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA, pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses, with which A375 cells were infected. Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein.</p><p><b>RESULTS</b>The lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group.</p><p><b>CONCLUSION</b>The lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.</p>