摘要
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。
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.
出处
《中国实验血液学杂志》
CAS
CSCD
2011年第6期1343-1347,共5页
Journal of Experimental Hematology
基金
国家自然科学基金重大项目
编号90919044
国家自然科学基金青年科学基金项目
编号81000221
国家自然科学基金面上项目
编号30971297
北京市科技新星计划
编号2010B075
