AKR1B10基因沉默对肝癌细胞增殖和凋亡的影响

目的探索肝细胞癌(HCC)中醛酮还原酶家族1成员B10(AKR1B10)的生物学功能和相关的病理机制。方法通过CCLE数据库分析正常肝细胞与HCC细胞系中AKR1B10 mRNA的表达,UALCAN以及Oncomine数据库分析癌旁组织与HCC组织中AKR1B10 mRNA的表达,Western Blot法验证HCC患者癌组织与癌旁组织中AKR1B10蛋白表达差异。使用慢病毒分别敲减HepG2及Huh7细胞中的AKR1B10,分为对照组和AKR1B10敲减组(shAKR1B101#、shAKR1B102#)。采用AnnexinV-APC/PI双染法、细胞克隆实验、皮下移植肿瘤的方法检测敲减AKR1B10后HCC细胞凋亡、克隆形成、皮下成瘤体积的变化。过氧化物敏感性荧光探针DCFH-DA以及Western Blot检测敲减AKR1B10后HCC细胞内活性氧(ROS)以及p-ATMser1981、γ-H2AX、c-Caspase-3和c-RARP等蛋白的变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK-q检验。结果CCLE数据库提示AKR1B10 mRNA在HCC细胞中的表达明显高于正常肝细胞;Oncomine数据库显示,与癌旁组织相比,HCC组织中AKR1B10 mRNA的表达增加了10倍以上;UALCAN数据库提示AKR1B10基因在HCC组织中高表达;本研究的6例HCC患者中AKR1B10在HCC组织中的表达高于癌旁组织。与对照组相比,AKR1B10敲减组中HCC细胞的克隆形成能力明显下降(P值均<0.001),细胞凋亡率明显增加(P值均<0.001)。与对照组相比,AKR1B10敲减组ROS的水平升高,DNA损伤指标p-ATMser1981、γ-H2AX蛋白,细胞凋亡指标c-Caspase-3和c-RARP蛋白增加。此外,在BALB-c/Nude小鼠模型中,从异体移植后第13天开始,与对照组相比,AKR1B10敲减组肿瘤体积明显缩小(P值均<0.05)。结论AKR1B10可能是治疗HCC的有效治疗靶标。

Effect of Aldo-keto reductase family 1 member B10 gene silencing on the proliferation and apoptosis of hepatocellular carcinoma cells

Objective To investigate the biological functions and related pathological mechanisms of Aldo-keto reductase family 1 member B10(AKR1B10)in hepatocellular carcinoma(HCC).Methods The CCLE database was used to analyze the mRNA expression of AKR1B10 in normal liver cells and HCC cell lines.The UALCAN and Oncomine databases were used to analyze the mRNA expression of AKR1B10 in HCC tissue and adjacent tissue.Western blot was used to investigate the difference in the protein expression of AKR1B10 between HCC tissue and adjacent tissue.AKR1B10 in HepG2 and Huh7 cells was knocked down by lentivirus,and the cells were divided into control group and AKR1B10 knockdown group(which was further divided into shAKR1B101#group and shAKR1B102#group).AnnexinV-APC/PI double staining,colony formation assay,and the method of subcutaneous xenograft tumor were used to observe the apoptosis of HCC cells,colony formation,and the change in the volume of subcutaneous xenograft tumor after AKR1B10 knockdown.The peroxide-sensitive fluorescent probe DCFH-DA and Western blot were used to measure the changes in ROS and related proteins,such as p-ATMser1981,γ-H2AX,c-Caspase-3,and c-RARP in HCC cells.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the SNK-q test was used for further comparison between two groups.Results The CCLE database showed that the mRNA expression of AKR1B10 in HCC cells was significantly higher than that in normal liver cells;the Oncomine database suggested that the mRNA expression of AKR1B10 in HCC tissue was more than 10 times that in adjacent tissue;the UALCAN database showed that the AKR1B10 gene was highly expressed in HCC tissue.For the six patients in this study,the expression of AKR1B10 in HCC tissue was significantly higher than that in adjacent tissue.Compared with the control group,the AKR1B10 knockdown group had a significant reduction in the colony formation ability of HCC cells(P<0.001)and a significant increase in apoptosis rate(P<0.001).Compared with the control group,the AKR1B10 knockdown group had significant increases in ROS,the markers for DNA damage p-ATMser1981 andγ-H2AX,and the indices for cell apoptosis c-Caspase-3 and c-RARP.In the BALB-c/Nude mouse model,compared with the control group,the AKR1B10 knockdown group had a significant reduction in tumor volume since day 13 after allograft transplantation(P<0.05).ConclusionAKR1B10 may be an effective therapeutic target for the treatment of HCC.