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

摘要/Abstract

摘要： 目的：构建微小RNA-186（miR-186）基因的海绵载体，建立稳定敲减miR-186的EA.hy926细胞株。方法：化学合成miR-186海绵体序列，并克隆到慢病毒FV040表达载体上；采用lipofectamine 2000共转染FV040-miR-186-sponge载体和慢病毒辅助包装质粒到HEK293T细胞，包装慢病毒，测定病毒滴度；FV040-control慢病毒作为对照组，FV040-miR-186-sponge作为实验组，分别感染EA.hy926细胞，筛选稳转细胞株；采用荧光定量PCR （qPCR）法检测空白对照组、FV040-control对照组及FV040-miR-186-sponge实验组中EA.hy926细胞miR-186的相对表达水平。结果：测序结果显示克隆的目的基因序列与设计的miR-186海绵体序列完全一致。在感染的EA.hy926细胞中观察到绿色荧光蛋白（GFP）的表达。FV040-control包装的慢病毒滴度为2×10⁸ TU·mL^-1，FV040-miR-186-sponge组慢病毒滴度为6×10⁸ TU·mL^-1；成功建立了稳定表达miR-186-sponge的EA.hy926细胞株，感染效率达95%；qPCR检测，与空白对照组和FV040-control组比较，FV040-miR-186-sponge组EA.hy926细胞中miR-186相对表达水平降低（P<0.01）。结论：成功构建了miR-186基因的海绵载体，建立了稳定下调miR-186表达的EA.hy926-miR-186-sponge细胞株。

关键词: 微小RNA-186, 海绵体, 慢病毒, EA.hy926细胞

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

中图分类号:

R543.5

王梦旭, 李胜男, 胡伟东, 陈少凤, 陈杏兰, 李友. miR-186海绵载体的构建及其在EA.hy926细胞株中的表达[J]. 吉林大学学报(医学版), 2019, 45(03): 498-503.

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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