miR-186海绵载体的构建及其在EA.hy926细胞株中的表达

doi:10.13481/j.1671-587x.20190306

Abstract

Abstract: Objective:To construct the sponge vector which can target microRNA-186(miR-186),and to create a stable EA.hy926 cell 1ine that can knockdown miR-186. Methods:The miR-186 sponge sequence was chemically synthesized and cloned into lentiviral vector FV040. Then the FV040-miR-186-sponge recombinant plasmid together with the helper plasmids were cotransfected into the HEK293T cells by using lipofectamine 2000 to package lentivirus and the viral titer was determined. The FV040-control lentivirus (designated as the control group), and the FV040-miR-186-sponge (designated as the experiment group) were used to infect EA.hy926 cells for establishing stable cell lines.The fluorescent quantitative PCR (qPCR) method was used to detect the relative expression levels of miR-186 in the EA.hy926 cells in blank group,FV040-control group and FV040-miR-186 sponge group, respectively. Results:The cloned target sequence was identical with the designed miR-186 sponge sequence. The green fluorescence protein(GFP) was observed in the infected EA.hy926 cells. The lentivirus titre of viruses in the FV040-control group was 2×10⁸ TU·mL^-1, and which was 6×10⁸ TU·mL^-1 in FV040-miR-186 sponge group. The EA.hy926 cell line stably expressed miR-186-sponge was established successfully and the infection rate was as high as 95%. The qPCR results indicated that the relative expression level of miR-186 in the EA.hy926 cells in FV040-miR-186-sponge group was lower than those in blank control group and FV040-control group(P<0.01). Conclusion:The miR-186-sponge vector is successfully constructed, and the EA.hy926-miR-186-sponge cell line with the stably decreased expression of miR-186 is established successfully.

Key words: miR-186, sponge, lentivirus, EA.hy926 cell

CLC Number:

R543.5

WANG Mengxu, LI Shengnan, HU Weidong, CHEN Shaofeng, CHEN Xinglan, LI You. Construction of miR-186 sponge vectorand its expression in EA.hy926 cells[J].Journal of Jilin University(Medicine Edition), 2019, 45(03): 498-503.

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Construction of miR-186 sponge vectorand its expression in EA.hy926 cells

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