乏氧条件下组蛋白去乙酰化酶6对肝癌细胞增殖转移能力的影响

Effects of histone deacetylase 6 on the proliferation and metastasis of liver cancer cells under hypoxia

目的探讨乏氧条件下组蛋白去乙酰化酶6(HDAC6)对肝癌细胞增殖转移能力的影响。方法取46例肝癌患者的肝癌组织及相应的癌旁组织,采用实时荧光定量逆转录聚合酶链反应(RT-PCR)检测HDAC6 mRNA的相对表达量、免疫组化法检测HDAC6蛋白的表达情况。将转染HDAC6小干扰RNA(siRNA)的乏氧人肝癌HepG2细胞作为Hp-siHDAC6组,未转染的乏氧人肝癌HepG2细胞作为Hp组,将常氧人肝癌HepG2细胞作为NC组。采用定量RT-PCR法检测3组人肝癌HepG2细胞HDAC6 mRNA的相对表达量,采用CCK8法检测细胞增殖能力,Transwell实验检测细胞迁移、侵袭能力。结果肝癌患者肝癌组织中HDAC6 mRNA的相对表达量明显低于癌旁组织(P﹤0.01),HDAC6蛋白阳性表达率为34.78%,明显低于癌旁组织的73.91%(P﹤0.01)。肿瘤直径≥5 cm、术前甲胎蛋白(AFP)水平≥400 ng/ml、分化程度为低分化、TNM分期为Ⅲ+Ⅳ期的肝癌患者肝癌组织中HDAC6阳性表达率分别低于肿瘤直径﹤5 cm、术前AFP水平﹤400 ng/ml、分化程度为中高分化、TNM分期为Ⅰ+Ⅱ期的患者,差异均有统计学意义(P﹤0.05)。Hp组和Hp-siHDAC6组细胞中HDAC6 mRNA的相对表达量均明显低于NC组(P﹤0.01),Hp-siHDAC6组细胞中HDAC6 mRNA的相对表达量明显低于Hp组(P﹤0.01)。转染48、72、96 h后,Hp组和Hp-siHDAC6组细胞的光密度(OD)值均明显高于NC组(P﹤0.01),Hp-siHDAC6组细胞OD值均明显高于Hp组(P﹤0.01)。Hp组和Hp-siHDAC6组细胞迁移数、细胞侵袭数均明显高于NC组(P﹤0.01),Hp-siHDAC6组细胞迁移数、细胞侵袭数均明显高于Hp组(P﹤0.01)。结论HDAC6蛋白在肝癌组织中表达下调,与肝癌患者的肿瘤直径、术前AFP水平、分化程度、TNM分期密切相关。乏氧可增强人肝癌HepG2细胞的增殖迁移能力,其机制可能与HDAC6基因的表达下调有关。

Objective To investigate the effect of histone deacetylase 6(HDAC6)on the proliferation and metastasis of liver cancer cells under hypoxia.Method The liver cancer tissues and corresponding adjacent tissues of 46 patients with liver cancer were selected,and the relative expression of HDAC6 mRNA was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)method,and the expression of HDAC6 protein was detected by immunohistochemistry.Hypoxic human hepatoma HepG2 cells transfected with HDAC6 small interfer-ing RNA(siRNA)were used as the Hp-siHDAC6 group,non-transfected hypoxic human hepatoma HepG2 cells were used as the Hp group,and normoxic human hepatoma HepG2 cells were used as the NC group.Quantitative RT-PCR was used to detect the relative expression of HDAC6 mRNA in the three groups of human hepatoma HepG2 cells,CCK8 meth-od was used to detect the cell proliferation ability,and Transwell assay was used to detect the cell migration and invasion ability.Result The relative expression of HDAC6 mRNA in liver cancer tissues of patients with liver cancer was signifi-cantly lower than that in adjacent tissues(P<0.01),and the positive expression rate of HDAC6 protein was 34.78%,which was significantly lower than 73.91%in adjacent tissues(P<0.01).The positive expression rate of HDAC6 in hepa-tocellular carcinoma tissues of liver cancer patients with tumor diameter≥5 cm,preoperative alpha-fetoprotein(AFP)lev-el≥400 ng/ml,poorly differentiated,and TNM stage III+IV were lower than the patients with tumor diameter<5 cm,preoperative AFP level<400 ng/ml,moderate or high differentiation degree,and TNM stage I+II,and the differences were statistically significant(P<0.05).The relative expression of HDAC6 mRNA in Hp group and Hp-siHDAC6 group were significantly lower than those in NC group(P<0.01),and the relative expression of HDAC6 mRNA in Hp-siHDAC6 group were significantly lower than those in Hp group(P<0.01).After 48,72,and 96 h of transfection,the optical density(OD)value of cells in Hp group and Hp-siHDAC6 group were significantly higher than those in NC group(P<0.01),and the OD value of cells in Hp-siHDAC6 group was significantly higher than that in Hp group(P<0.01).The number of cell migration and cell invasion in Hp group and Hp-siHDAC6 group were significantly higher than those in NC group(P<0.01),and the number of cell migration and cell invasion in Hp-siHDAC6 group were higher than those in Hp group(P<0.01).Conclusion The expression of HDAC6 protein is down-regulated in liver cancer tissue,and it is closely related to the tumor diameter,preoperative AFP level,differentiation degree and TNM stage of liver cancer.Hypoxia can enhance the proliferation and migration ability of human hepatoma HepG2 cells,and the mechanism may be related to the down regulation of HDAC6 gene expression.