miR-373-3p低表达慢病毒载体构建及其对子宫内膜癌细胞增殖迁移的抑制作用

发布时间：2018-07-07 12:03

本文选题：子宫内膜癌 + HEC-A细胞　；参考：《山东医药》2017年45期

【摘要】：目的构建人miR-373-3p低表达慢病毒载体,探讨miR-373-3p低表达对子宫内膜癌HEC-1A细胞增殖和迁移能力的影响。方法利用PCR扩增包含miR-373-3p前体序列的目的基因,经酶切后克隆入慢病毒载体GV280中构建GV280-pre-miR-373-3p inhibitor表达载体,通过与包装质粒共转染293T细胞,进行慢病毒的包装和滴度测定。分别用空载慢病毒(空载病毒组)及重组慢病毒miR-373-3p(miR-373-3p组)感染HEC-1A细胞,并设立空白对照组。采用RT-PCR法检测细胞miR-373-3p mRNA的相对表达水平,CCK-8法检测培养24、48、72、96 h时细胞增殖活力,细胞划痕和Transwell小室试验检测培养24、48 h时细胞迁移能力。结果成功构建了人miR-373-3p低表达慢病毒载体。与空白对照组和空载病毒组比较,miR-373-3p组miR-373-3p相对表达量低,培养48、72、96h HEC-1A细胞增殖活力均降低(P均0.05),24、48 h HEC-1A细胞划痕面积迁移率低,24 h穿膜细胞数少(P均0.05)。空载病毒组及空白对照组各指标比较,P均0.05。结论成功构建了人miR-373-3p低表达慢病毒载体,miR-373-3p低表达可抑制人子宫内膜癌细胞HEC-1A的增殖、迁移能力。
[Abstract]:Objective to construct a low expression lentivirus vector of human miR-373-3p and to investigate the effect of low expression of miR-373-3p on the proliferation and migration of endometrial carcinoma HEC-1A cells. Methods the target gene containing the precursor sequence of miR-373-3p was amplified by PCR and cloned into the lentivirus vector GV280. The GV280-pre-miR-373-3p inhibitor expression vector was constructed and cotransfected with the packaging plasmid 293T cells for the packaging and titer determination of lentivirus. HEC-1A cells were infected with no-load lentivirus (no-load virus group) and recombinant lentivirus miR-373-3p (miR-373-3p group), respectively. The relative expression level of miR-373-3p mRNA was detected by RT-PCR and the cell proliferation activity was detected by CCK-8 method at 24: 48 / 722 / 96 h, and the cell migration ability was detected by cell scratch and Transwell chamber test at 24: 48 h. Results the low expression lentiviral vector of human miR-373-3p was successfully constructed. The relative expression of miR-373-3p in miR-373-3p group was lower than that in blank control group and no-load virus group, and the proliferative activity of HEC-1A cells was decreased (P 0.05). The migration rate of scratch area of HEC-1A cells at 244h was lower than that of HEC-1A cells at 24 h (P 0.05). The comparison of each index between the no-load virus group and the blank control group (P < 0.05). Conclusion the low expression of human miR-373-3p can inhibit the proliferation and migration of human endometrial cancer cell line HEC-1A.
【作者单位】：广西医科大学第一附属医院;
【基金】：广西医疗卫生适宜技术研究与开发项目(S201520)
【分类号】：R737.33

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