HIF-1α反义寡核苷酸对肝癌细胞增殖凋亡的影响及机制

Effects and it's mechanism of HIF-1α ASODN on the cell proliferation and the apoptosis of human hepatocellular carcinoma cell

目的本研究通过应用HIF-1α反义寡核苷酸(antisense oligodeoxynucleotide,ASODN)处理SMMC-7721肝癌细胞,观察其对SMMC-7721肝癌细胞增殖的作用和对细胞周期及凋亡率的影响及其机制。方法肝癌细胞系SMMC-7721分对照组和试验组。对照组细胞常规培养,试验组细胞施加HIF-1α的ASODN干预,设浓度梯度和时间梯度。检测不同药物浓度、不同时间对SMMC-7721细胞的增殖的作用及对细胞周期和凋亡率的影响。对bcl-2、bax、surviving mRNA表达产物相对定量,并对SMMC-7721细胞胞浆bcl-2、bax、survivin蛋白进行相对定量分析。结果与对照组相比,各处理组OD值均有显著下降,细胞数量降低,且各实验组相比呈明显的时间-剂量依赖效应,差异有统计学意义(P<0.05)。不同浓度HIF-1α的ASODN处理细胞后,均可使各处理组G0/G1期细胞增多,S期细胞减少,且呈时间-剂量依赖效应,差异有统计学意义(P<0.05)。不同浓度HIF-1α的ASODN处理细胞不同时间后,与对照组相比,各实验组bcl-2 mRNA和survivin mRNA表达均有显著下降,差异有统计学意义(P<0.05)。而bax的mRNA无明显变化趋势(P>0.05)。不同浓度HIF-1α的ASODN处理细胞不同时间后,与对照组相比,各实验组bcl-2和survivin的蛋白表达显著下降,bax的蛋白表达显著增加,差异有统计学意义(P<0.05)。结论 HIF-1α的ASODN可以将SMMC-7721肝癌细胞株阻滞于G0/G1期,影响细胞周期进程,抑制细胞增殖,促进细胞凋亡。HIF-1α促进增殖抑制凋亡的可能机制是上调肝癌细胞Survivin和bcl-2的表达,下调bax的表达。针对HIF-1α抑制剂的应用为肝癌的治疗提供了新思路。

Objective To explore the effects and mechanism of HIF-1α ASODN on the cell proliferation and the apoptosis of human hepatocellular carcinoma cell line SMMC-7721 and it＇s mechanism. Methods SMMC-7721 cells were divided as control group and experiment group. The former were cultured with normal condition,the later were treated by HIF-1α ASODN with various concentration and time. Changes of cell cycle and apoptosis rate after treated by HIF-1α ASODN with various concentration and time were detected. The expression of bcl-2,bax,survivin mRNA level in cultured SMMC-7721 was evaluated by RT-PCR. The bcl-2,bax,survivin protein in cytoplasm was evaluated by western blot. Results After treated with HIF-1α ASODN,the OD values of treated groups decreased compared with control group,and there was statistically significant difference between control group and every treatment group（ P〈0. 05）. The inhibition of the proliferation of SMMC-7721 cells by HIF-1α ASODN displays a dose-and-time-dependent manner（ P〈0. 05）. The distribution of cell cycle by flow cytometry indicates that after treated with HIF-1α ASODN,the number of cells in G0 / G1 phase increased gradually,while the number of cells in S phase decreased gradually,which was in a dose-and-time- dependent manner. Following the increasing of the concentration of HIF-1α ASODN,the expression of bcl-2 mRNA and survivin mRNA decreased gradually compared with control group. But there was no obvious change of the bax mRNA（ P〈0. 05）. Following the increasing of the concentration of HIF-1α ASODN,expression of bcl-2 protein and survivin protein decreased gradually,but bax protein increased gradually. Conclusion The HIF-1α ASODN can inhibit the proliferation and affect the cell cycle by inhibiting the G1 period of SMMC-7721 cell. The mechanisms were supposed to be that HIF-1α up-regulates the expression of Survivin and bcl-2 and down-regulates the expression of bax. The effects were in time-dependent pattern. The application of the HIF-1α inhibitor offer a new way for the treatment of the HCC.