14-3-3ξ对HL-60和HL-60/VCR细胞增殖的抑制作用

摘要

This study was aimed to investigate the expression and role of 14-3-3ξ in the AML cell lines:sensitive HL-60 and drug-resistant HL-60/VCR cells.Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray.Western blot was performed to investigate the expression of Pgp,14-3-3ξ,and anti-apoptosic protein BCL-2,MCL-1 proteins.Immunofluorescence assay was used to detect the subcellular location of 14-3-3ξ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy.Transduction with siRNA was used to silence 14-3-3ξ in AML cell lines.Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells.The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells,but significantly overexpressed in HL-60/VCR cells.Except 14-3-3（o）,the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells,especially 14-3-3ξ.The higher expression of 14-3-3ξ,BCL-2,MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells.These results were same results from gene chip.It was also noticed that 14-3-3ξ was located in the cytoplasma and nuclei of AML cell lines,especially over-expressed in HL-60/VCR cells.Furthermore,suppression of 14-3-3ξ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1,especially in HL-60/VCR cells.It is concluded that 14-3-3ξ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression.These results suggested that development of therapy targeting 14-3-3ξ may provide novel,effective strategies for refractory and relapsed AML.%本研究旨在进一步探讨14-3-3ξ在急性髓系白血病(acute myeloid leukemia,AML)敏感细胞HL-60和耐药细胞HL-60/VCR中的表达和作用.用半定量RT-PCR和Western blot分别检测多药耐药基因mdr1 mRNA和Pgp的表达以验证基因芯片的结果,用Western blot检测PgP、14-3-3ξ以及抗凋亡分子BCL-2、MCL-1蛋白的表达;免疫荧光法和激光共聚焦法检测14-3-3ξ在HL-60和HL-60/VCR亚细胞中的定位.采用将小干扰RNA(siRNA)通过脂质体LipofeetamineTM 2000介导,瞬时转染HL-60和HL-60/VCR细胞,以观察细胞生长的变化.细胞增殖指标采用生长曲线和细胞周期测定法测定.采用RT-PCR和Western blot方法检测PgP、14-3-3ξ以及抗凋亡分子BCL-2、MCL-1表达的变化.结果表明,mdr1 mRNA和Pgp仅在HL-60/VCR细胞中高表达.与HL-60细胞相比,除了14-3-3(α)之外,其他14-3-3亚型均在HL-60/VCR细胞中高表达,尤其是14-3-3ξ同时伴有BCL-2和MCL-1高表达.另外,研究发现14-3-3ξ主要定位于细胞浆和核.通过RNA干涉抑制14-3-3ξ的表达能抑制HL-60和HL-60/VCR细胞的生长,下调BCL-2和MCL-1蛋白.结论:14-3-3ξ可能通过BCL-2和MCL-1介导调控HL-60和HL-60/VCR细胞的增殖,是治疗难治复发AML的潜在新靶点.