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  • Title: [Construction and package of a recombinant adenoviral vector to inhibit miR-21 expression and its effects on HepG2 cells in vitro].
    Author: Zhang YL, Tang JZ, Liu HP, Zhang ZB, Xu WJ, Wei J, Xu GX.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2012 Dec; 28(12):1265-8. PubMed ID: 23232518.
    Abstract:
    AIM: To construct the recombinant adenoviral vector which can efficiently inhibit mature miR-21 expression and explore its effects and the underlying mechanisms on hepatoma cell line HepG2. METHODS: Based on miRNA-sponge technology, we synthesized 8 duplicated fragments, fully complementary with the miR-21 sequence and cloned them into a shuttle vector pAdTrack-CMV. The constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria, then transfected into 293A cells. The recombinant adenovirus was used to challenge HepG2 cells. Mature miR-21 level was detected by real-time PCR. Apoptosis and proliferation of the HepG2 cells were detected by Hochest 33258 staining, Western blotting and MTT assay. RESULTS: The restriction enzyme digestion, DNA sequencing and detection of GFP expression demonstrated that recombinant adenoviral vector was constructed successfully. The recombinant adenovirus inhibited the expression of miR-21 in HepG2 cells, and also depressed the proliferation of HepG2 cells and promoted the apoptosis. CONCLUSION: The recombinant adenoviral vector we successfully constructed could efficiently reduce the expression of miR-21 in HepG2 cells and depress the proliferation of HepG2 cells.
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