miR-128真核表达载体的构建及功能的初步研究

目的构建miR-128真核表达载体,研究其在神经胶质瘤细胞株U343表达及对U343细胞增殖的影响。方法以人神经胶质瘤细胞株U343的DNA为模板扩增miR-128-1和miR-128-2的前体序列,将人miR-128-1(hsamiR128-1)和人miR-128-2(hsa-miR128-2)基因克隆到真核表达载体pCR3.1,将pCR3.1-miR-128-1(p-128-1)和pCR3.1-miR-128-2(p-128-2)表达载体瞬时转染U343细胞,采用Northern印迹检测成熟的miR-128表达。将U343细胞接种96孔板,MTT法检测过表达miR-128对细胞增殖的影响。结果p-128-1和p-128-2表达载体经酶切及测序鉴定正确;二者转染细胞后,反转录-聚合酶链反应(RT-PCR)扩增结果显示,其能有效表达miR-128的前体;Northern印迹结果均能产生成熟态的miR-128。MTT检测结果在无血清培养和维甲酸处理的情况下,细胞的增殖能力明显下降。结论成功构建了p-128-1和p-128-2的真核表达载体,转染神经胶质瘤细胞后能有效表达,miR-128基因过表达能抑制U343细胞的增殖。

Construction of eukaryotic expression vector miR-128 and preliminary study on its function

Objective： To construct a recombinant miR-128 eukaryotic expression vector and express it in human glioma cells U343 for detecting its effect on the proliferation of U343 cells. Methods： The sequences of pre-miR-128-1 and pre-miR-128-2 were amplified by PCR from human genomic DNA, and then inserted into pCR3.1 vector to generate pCR3.1-miR-128-1 （p-128-1） and pCR 3.1-miR-128-2 （p-128-2） which was transfected into human glioma cells U343. The expression of miR-128 was verified by RT-PCR and Northern blotting. The effect of p-128-1 or p-128-2 on U343 cell proliferation was detected by MTT method. Results： p-128-1 and p-128-2 expression vectors turned out to be correct by digesting and DNA sequencing. RT-PCR results indicated that miR-128 precursors were effectively expressed in U343 cells transfected by p-128-1 and p-128-2. Northern blotting results showed that mature miR-128 can be produced in U343 cells transfected by p-128-1 or p-128-2. MIT results showed that overexpression of mir-128 can reduce the proliferation of U343 cells. Conclusions： The eukaryotic expression vectors p-128-1 and p-128-2 are successfully constructed and effectively express in U343 cells. The overexpression of miR-128 inhibits human glioma cells U343 proliferation.