HIF-1α反义寡核苷酸对肝癌细胞增殖凋亡的影响及机制

摘要

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.%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。