miR-328通过C/EBPα表达上调抑制K562细胞增殖

miR-328 Inhibits K562 Cell Proliferation by Up-regulation of C/EBPa Expression

目的:本研究旨在探讨miR-328(microRNA-328)对慢性髓系白血病(CML)急变期细胞株K562增殖的影响及C/EBPα的介导作用。方法:构建miR-328靶向基因及抑制基因的真核表达载体hsa-miR-328及hsa-miR-328-inhibitor,并分别通过脂质体lipofectamineTM2000介导转染至K562细胞;通过荧光显微镜观察细胞转染情况;通过实时荧光定量RT-PCR检测miR-328、C/EBP mRNA的表达水平;Western-Blot方法测定C/EBPα蛋白的表达;CCK-8检测细胞增殖的情况。结果:本研究通过成功构建重组真核表达载体hsa-miR-328、hsa-miR-328-inhibitor;将载体转染K562细胞,24 h后在荧光显微镜下可见荧光细胞,表明载体成功转入K562细胞,48 h和72 h后可见荧光细胞数逐渐增多。实时荧光定量RT-PCR证实miR-328在K562细胞中成功表达,而miR-328对细胞C/EBPαmRNA表达无明显影响;通过Western blot检测发现miR-328恢复C/EBPα蛋白表达;细胞活力检测提示miR-328抑制K562细胞的增殖。结论:成功构建并转染miR-328基因至K562细胞,miR-328通过恢复C/EBPα的表达抑制K562细胞的增殖。

Objective： Our research was aim to investigate the effect of microRNA-328 （ miR-328 ） on proliferation of chronic myeloid leukemia （CML） cell K562 and the mediated effect of C/EBPa. Methods： The eukaryotic expression vectors of miR-328 targeting gene and suppressor gene （hsa-miR-328 and hsa-miR-328-inhibitor） were constructed, and transfected into K562 cells respectively. The mRNA expression levels of miR-328 and C/EBP a were detected by real-time fluorescence quantitative RT-PCR; C/EBP et protein expression was detected by Western blot; CCK-8 was used to estimate the cell viability. Results： The recombinant genes of hsa-miR-328 and hsa-miR-328- inhibitor were successfully constructed and transfected into K562 cells. Fluorescent cells were observed after 24 h, and the visible fluorescence cells were gradually increased after 48 h or 72 h, the miR-328 showed no effect on the mRNA expression of C/EBPa detected by RT-PCR. Meanwhile, miR-328 showed recovering effect on C/EBPa translation and inhibition of K562 cells proliferation. Conclusion： miR-328 has been successfully constructed and transfected into K562 cells, miR-328 inhibits the proliferation of K562 cells by up-regulation of C/EBP α.