Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7

doi:10.13560/j.cnki.biotech.bull.1985.2015.05.035

Abstract

Abstract: This study aimed to construct the lentiviral RNA interference（RNAi）vector of human FUT8 gene and determine its effect on proliferation of human breast cancer cells MCF-7. Designing three short hairpin RNA（shRNA）sequence targeting the FUT8 gene, and then synthesizing the complementary DNA chains containing the target sequence, miRNA lentiviral vector plasmid with linear pGC-LV-GFP carriers were constructed and then transformed to DH5α cells. After identified by sequencing, then packaging lentiviral vectors and measuring the virus titer were done. The recombinant lentiviral vector pGC-shFUT8 was transfected into human MCF-7 cells, and then the expression of FUT8 in transfected MCF-7 cells was detected by real time-PCR and Western Blotting. The effect of shFUT8 on MCL-7 cell proliferation was measured by MTT assay and colony formation experiment. Gene sequencing confirmed the successful construction of RNAi lentiviral vector targeting the FUT8 gene. The lentiviral vectors were packed successfully by 293T cells and the virus titer was more than 5×10⁸TU/mL. Expressed GFP in transfected cells were observed under the fluorescence microscope, and the transfection efficiency was over 90%. Real-time PCR and Western Blotting analysis indicated that the expression levels of FUT8 mRNA and protein in the transfected group significantly reduced when comparing with the negative control group. Particularly the pGC-shFUT8-2 sequence could interfere 80 % of FUT8 gene expression. The proliferation of MCF-7 decreased after FUT8 was inactive.

Key words: FUT8 gene, Lentiviral vector, RNA interference, MCF-7cells

Wen Xianchun, Han Cuicui, Zhao Yuesheng, Yu Haitao, Li Chengchong, Yue Liling. Construction of FUT8 Gene Lentiviral RNA Interference Vector and Regulation on Proliferation of Human Breast Cancer Cells MCF-7[J]. Biotechnology Bulletin, 2015, 31(5): 231-236.

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