MicroRNA-193b对K562细胞C-KIT蛋白表达及生物学行为的影响

本研究探讨microRNA-193b（miR-193b）对K562白血病细胞系C-KIT蛋白表达及生物学行为的影响。采用HiPerFect转染试剂介导FAM荧光标记的miR-193b mimic或阴性对照分别转染过表达C-KIT的K562细胞系,采用流式细胞术检测FAM阳性细胞百分比以监测转染效率,采用Western blot检测C-KIT和磷酸化C-KIT蛋白表达水平,采用CCK-8试剂检测细胞生长曲线,采用AnnexinⅤ染色后流式细胞仪检测细胞凋亡,采用流式细胞仪检测粒系分化标志CD11b和CD15阳性细胞百分比。结果显示,miR-193b或阴性对照转染的K562细胞中,FAM阳性细胞可达80%左右;与对照组相比,过表达miR-193b可明显抑制K562细胞中C-KIT、磷酸化C-KIT蛋白表达水平,同时,K562细胞的增殖受到抑制,凋亡细胞、CD11b阳性细胞、CD15阳性细胞的百分比均有增加。结论：miR-193b可抑制K562细胞C-KIT表达,并抑制细胞增殖;该增殖抑制作用可能与miR-193b诱导的细胞凋亡、分化有关,本研究为进一步分析miR-193b在白血病发生中的作用提供了实验依据。

Effect of microRNA-193b on C-KIT protein expression and biological behaviors of K562 cells

The aim of this study was to investigate the effect of microRNA-193b (miR-193b) on C-KIT protein expression and biological behaviors in K562 cells. The FAM-labeled miR-193b mimic and negative control were respectively transfected into K562 cells using HiPerFect transfection reagent. The percentage of FAM-positive cells was monitored by flow cytometry. The levels of C-KIT and phosphorylated C-KIT protein were detected by Western blot. The cell growth was measured by CCK-8 reagent. The apoptosis of cells were analyzed by flow cytometry with Annexin V staining. The differentiation of cells were analyzed by flow cytometry with anti-CD11b or anti-CD15 staining. The results demonstrated that the percentage of FAM-positive cells was about 80% in miR-193b or negative control-transfected K562 cells. Compared with the negative control group, overexpression of miR-193b in K562 cells significantly inhibited the levels of C-KIT and phosphorylated C-KIT protein. Meanwhile, the cell growth was inhibited and the percentages of apoptotic cells, CD11b- or CD15-positive cells increased. It is concluded that miR-193b can reduce C-KIT expression and inhibit cell growth in K562 cells. The growth-inhibitory activity of miR-193b is associated with apoptosis and granulocytic differentiation. This study contributed to further investigate the role of miR-193b in leukemogenesis.