HINT1对人黑素瘤细胞A375增殖和凋亡的影响

目的 探讨HINTl基因对人黑素瘤细胞A375增殖、凋亡的影响及其作用机制.方法 应用构建的peDNA.3.1/mye-His(-)A-HINTl真核表达载体,建立稳定转染且能高表达HINTI的A375细胞模型.用M1rr法检测细胞增殖活性变化,流式细胞仪检测细胞周期及细胞凋亡率,TUNEL法检测细胞凋亡.分光光度法检测Caspase 3,Caspase 8及Caspase 9的相对活性.Western印迹法检测bcl-2、bax、细胞色素C及p53的表达.结果 与空载体-A375及未转染A375细胞相比,HINT1-A375细胞生长速度明显减慢(P<0.05);Gl期细胞增多(73.17%±3.99%,F=25.65,P<0.05),S期细胞减少(16.75%±1.62%.F=75.48,P<0.01),细胞出现明显的晚期凋亡(23.57%±9.58%,F=11.71,P<0.01). TUNEL法显示凋亡阳性细胞百分比(12%±1%)显著增高(F=358.02,P<0.01);Caspase 9(0.45±0.03,F=135.62,P<0.01)和Caspase 3表达(0.46±0.04,F=90.28,P<0.01)升高.凋亡相关蛋白bax、细胞色素C及p53表达增加,bel-2表达降低.结论 高表达H1NT1可抑制A375细胞的增殖,促进其凋亡,细胞周期出现G_1期阻滞,提示HINT1可能是人黑素瘤的抑癌基因.

Effects of histidine triad nucleotide-binding protein 1(HINT1)on the proliferation and apoptosis of human melanoma cell line A375

Objective To explore the effects of HINT1 on the proliferation and apoptosis of human melanoma cell line A375.Methods Three cell clones were used in the experiment.including A375 cells that were previously transfected with eukaryotic expressing vector pcDNA.3.1/myc-His(-)A-HINT1 and highly expressed HINTI(HINT1-A375),A375 cells transfected with pcDNA.3.1/myc-His(-)A empty vector(neo-A375)and untransfected A375 cells.M3T assay,flow cytometry and terminal deoxynucleotjdyl transferase (TdT)-mediated dUTP nick end labeling(TUNEL)were performed to detect the proliferation,cell cycle and apoptosis of the three kinds of cells,respectively.The relative activity of Caspase 3,8 and 9 was measured by spectrophotometry,and the protein expression of Bcl-2,Bax,Cytoehrome C and p53 by Western blot. Results Compared with neo-A375 and untransfected A375 cells,HINT1-A375 cells grew more slowly(P<0.05)with an increase in G1-phase population(73.17%±3.99%,F=25.65,P<0.05).a decrease in S-phase population(16.75%±1.62%,F=75.48,P<0.01),and a pronounced late apoptosis(23.57%±9.58%,F=11.71,P<0.01).A significant increase was also observed in the percentage of apoptotic cells(12%±1%,F=358.02,P<0.01)as well as in the relative activity of Caspase 9(0.45±0.03,F=135.62,P<0.01)and Caspase 3(0.46±0.04,F=90.28,P<0.01)in HINT1-A375 cells compared with the other two kinds of cells.Western blot showed upregulated expressions of Bax,Cytochrome C and p53 but downregulated expres-sion of Bel-2 in HINT1-A375 cells.Conclusions The overexpression of HINT1 could inhibit the proliferation and promote the apoptosis of A375 cells with cell cycle arrest in G1 phase,hinting that HINT1 may be a tumor suppressor gene in human melanoma.