鸟氨酸脱羧酶抗酶抑制因子-1高表达对小鼠黑素瘤B16-F1细胞增殖的影响

Effect of ornithine decarboxylase antizyme inhibitor-1 overexpression on the proliferation of B16-F1 mouse melanoma cells

目的:研究鸟氨酸脱羧酶抗酶抑制因子-1(ornithine decarboxylase antizyme inhibitor-1,OAZI-1)高表达对小鼠黑素瘤B16-F1细胞增殖的影响。方法:采用巢氏PCR法从小鼠肝癌H22细胞cDNA中克隆出小鼠OAZI-1基因,并构建重组质粒pcDNA3.1(+)/OAZI-1。采用脂质体转染法将重组质粒转染入B16-F1细胞后,Western印迹法和实时荧光定量PCR法鉴定高表达OAZI-1的B16-F1细胞株。MTT法和FCM检测OAZI-1高表达对B16-F1细胞增殖和细胞周期的影响。反相高效液相色谱检测OAZI-1高表达对细胞内多胺水平的影响。化学发光法检测细胞内精胺氧化酶(spermine oxidase,SMO)的活性。结果:成功获得高表达OAZI-1的B16-F1细胞克隆株B16/over,该细胞中OAZI-1在mRNA水平的表达量是转染空载体质粒对照组细胞B16/3.1的3.6倍。B16/over细胞中,鸟氨酸脱羧酶抗酶(ornithine decarboxylase antizyme,OAZ)的mRNA表达水平降低,鸟氨酸脱羧酶(ornithine decarboxylase,ODC)的mRNA表达水平升高。高表达OAZI-1能促进B16-F1细胞增殖,并影响细胞周期,G0/G1期细胞减少而S期和G2/M期细胞增加。B16/over细胞中腐胺的含量明显增加,精脒和精胺的含量减少,而且SMO活性升高。结论:OAZI-1高表达可能通过增加细胞内腐胺含量,促进黑素瘤细胞增殖,提示该抑制蛋白可能成为黑素瘤治疗的一个潜在靶点。

Objective：To study the effect of ornithine decarboxylase antizyme inhibitor-1（OAZI-1） overexpression on the proliferation of B16-F1 mouse melanoma cells.Methods：OAZI-1 gene was cloned out from the cDNAs derived from H22 mouse hepatocellular carcinoma cells,and then subcloned into the eukaryotic expression vector pcDNA3.1（＋）.The recombinant plasmid pcDNA3.1（＋）/OAZI-1 was transfected into B16-F1 cells with LipofectAMINE2000 reagents.The positive clone with OAZI-1 overexpression was identified by Western blotting and real-time fluorescent quantitative PCR.The effects of OAZI-1 overexpression on the cell proliferation and the cell cycle distribution were detected by MTT and flow cytometry.The level of polyamine was detected by reversed-phase high performance liquid chromatography.The chemiluminescence analysis was used to determine the activity of spermine oxidase（SMO）.Results：The B16-F1 clone with OAZI-1 overexpression was obtained successfully and named as B16/over.The expression level of OAZI-1 mRNA was 3.6-fold higher in the B16/over cells than that in the control B16/3.1 cells transfected with pcDNA3.1（＋）.The level of ornithine decarboxylase antizyme（OAZ） mRNA was decreased,and the level of ornithine decarboxylase（ODC） mRNA was elevated in B16/over cells.Overexpression of OAZI-1 in B16-F1 cells could promote the cell proliferation and influence the cell cycle distribution,including decreasing the cell number in G0/G1 phase and increasing the cell number in S and G2/M phases.In B16/over cells,the content of putrescine was increased,but the contents of spermidine and spermine were decreased,and the activity of SMO was elevated.Conclusion：Overexpression of OAZI-1 may promote the proliferation of B16-F1 cells by elevating the content of putrescine,which indicates that OAZI-1 may be a potential target for melanoma therapy.