Skp2反义寡核苷酸对K562细胞生长和增殖的影响

背景与目的:泛素-蛋白酶体途径是真核细胞中选择性降解短寿命蛋白的重要途径。S期激酶相关蛋白2(S-phasekinase-associatedprotein2,Skp2)是此途径中的一个重要组件,它是决定细胞周期素依赖性激酶抑制因子p27降解的关键蛋白。近年来,在实体瘤的研究中发现Skp2是癌蛋白,能促进癌细胞的细胞周期进展,但在白血病细胞恶性增殖中所起的作用还未见报道。本实验旨在研究Skp2反义寡核苷酸(antisenseoligodeoxynucleotides,ASODN)对白血病细胞系K562细胞生长和增殖的影响及其可能的机制。方法:Skp2反义寡核苷酸和K562细胞共同培养,通过显微镜下观察、四甲基偶氮唑蓝(MTT)实验观察K562细胞生长和增殖,流式细胞术分析细胞周期,逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)和细胞免疫化学测定Skp2mRNA、蛋白及p27mRNA、蛋白的表达。结果:Skp2反义寡核苷酸处理后,K562细胞增殖受抑,细胞周期阻滞于G0/G1期犤Skp2ASODN组(39.7±9.1)%,对照组(31.5±7.3)%,P<0.05犦。Skp2mRNA及Skp2蛋白水平降低;尽管p27mRNA未见改变,但其蛋白水平明显升高。结论:Skp2反义寡核苷酸能抑制K562细胞增殖,这种抑制作用主要是通过调控泛素-蛋白酶体途径,进而影响细胞周期进展而实现的。

Effect of Skp2 antisense oligodeoxynucleotide on growth and proliferation of K562 cells

BACKGROUND&OBJECTIVE:The ubiquitin-proteosome pathway is important for selective degradation of short-lived protein in eukaryotic cells. In this pathway Skp2 (S-phase kinase-associated protein 2) plays a critical role in degrading cyclin-dependent kinase inhibitor p27. It is verified that Skp2 is an oncoprotein that promotes cell cycle progression in solid tumor, but its role in leukemia cells remains unclear. This study was designed to investigate the effect of Skp2 antisense oligodeoxynucleotides(ASODN) on the growth and proliferation of leukemic K562 cells and its probable mechanism. METHODS: K562 cells were cocultured with Skp2 ASODN. The growth and proliferation of K562 cells were observed with light microscopy and MTT assay. The cell cycle was analyzed using flow cytometry. The expression levels of mRNA and protein were determined using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry, respectively. RESULTS: After treatment with Skp2 ASODN, the growth and proliferation of K562 cells were inhibited and the cell cycle was arrested at G(0)/G(1) phase(Skp2 ASODN group 39.7+/-9.1% vs. control group 31.5+/-7.3%,P< 0.05). Both Skp2 mRNA and its protein levels were down-regulated. Although p27 mRNA level remained unchanged, its protein level was up-regulated. CONCLUSION: Skp2 ASODN can inhibit the growth and proliferation of K562 cells, which is mediated by interfering with ubiquitin-proteosome system and regulating of cell cycle progression.