siRNA沉默RALA基因影响人白血病K562细胞增殖和凋亡

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

目的:研究小干扰RNA(siRNA)抑制v-ral猴白血病病毒癌基因同系物A(RALA)基因表达对人慢性粒细胞性白血病K562细胞增殖和凋亡的影响。方法:利用LipofectamineTM2000将化学合成的RALA siRNA转染体外培养的K562细胞,四甲基偶氮唑蓝(MTT)法检测RALA siRNA对K562细胞增殖的影响;台盼蓝拒染法检测RALA siRNA对K562细胞存活率的影响;real-time PCR检测RALA mRNA表达水平;Western blotting检测RALA蛋白表达水平;annexin V/PI双染流式细胞仪检测RALA siRNA对K562细胞凋亡的影响;Hoechst 33258染色荧光显微镜观察细胞形态学变化。结果:RALA siRNA可明显抑制K562细胞内RALA mRNA和蛋白的表达(P<0.05);与阴性对照组相比,转染RALA siRNA的K562细胞增殖明显受到抑制(P<0.05);流式细胞术结果显示转染RALA siRNA的K562细胞凋亡较阴性对照组显著增加(P<0.05);Hoechst 33258染色见转染RALA siRNA的K562细胞出现典型的凋亡形态学变化。结论:癌基因RALA在白血病发生发展过程中发挥重要作用。siRNA下调RALAmRNA和蛋白的表达,可抑制人白血病K562细胞增殖,诱导细胞凋亡,提示RALA可能是白血病治疗的新靶点。

AIM： To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A（RALA） on proliferation and apoptosis of chronic myelogenous leukemia（CML） K562 cells.METHODS： The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using LipofectamineTM 2000.The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion.The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively.The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide,and the apoptotic morphological changes were detected by Hoechst 33258 staining.RESULTS： RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells（P0.05）.The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group（P0.05）.The apoptotic rate was much higher in RALA siRNA group than that in negative control group（P0.05）.The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA.CONCLUSION： The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells,indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.