[Construction of a lentiviral RNA interference system targeting heparanase based on miR30 and its silencing effect].
Zhejiang Da Xue Xue Bao Yi Xue Ban
; 42(1): 67-74, 2013 01.
Article
em Zh
| MEDLINE
| ID: mdl-23505110
ABSTRACT
OBJECTIVE:
To construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.METHODS:
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.RESULTS:
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.CONCLUSION:
The lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.
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Base de dados:
MEDLINE
Assunto principal:
Lentivirus
/
MicroRNAs
/
RNA Interferente Pequeno
/
Vetores Genéticos
/
Glucuronidase
Idioma:
Zh
Ano de publicação:
2013
Tipo de documento:
Article