肾型谷氨酰胺酶在儿童肝母细胞瘤中的表达及作用研究

Expression profiles and roles of kidney-type glutaminase in hepatoblastoma

目的探究肾型谷氨酰胺酶(kidney-type glutaminase,KGA)在肝母细胞瘤中表达及与患儿预后的关系,探究KGA对肝母细胞瘤细胞增殖的影响。方法从GEO数据库获取44例肝母细胞瘤患儿的临床资料和测序数据,差异基因表达分析探究基因KGA在正常肝组织和肝母细胞瘤组织中的表达情况。根据KGA的中位mRNA相对水平表达量将44例患儿分为两组,即KGA高表达组(mRNA相对水平>37.181)和低表达组(mRNA相对水平<37.181),用Kaplan-Meier曲线比较两组患儿的生存差异。用单因素和多因素Cox回归分析研究基因KGA与患儿预后的关系。蛋白质印迹法检测KGA在正常肝细胞株LO2以及肝母细胞瘤细胞株HuH-6及HepG2中表达情况。分别用干扰小RNA和KGA抑制剂1(glutaminase-IN-1,GIN-1)处理HuH-6细胞,通过实时定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)和蛋白质印迹法检测KGA的mRNA相对表达水平和蛋白表达水平。细胞增殖能力检测(CCK8实验)和平板克隆形成实验检测细胞增殖能力变化。结果与正常肝脏组织及LO2细胞株相比,KGA在肝母细胞瘤中表达量显著升高。Kaplan-Meier生存分析显示KGA高表达组患儿无进展生存期更短(P=0.014)。多因素Cox回归分析表明KGA是肝母细胞瘤患儿预后的独立预测因子[HR(95%CI)为5.74(1.21~27.33),P=0.028]。RT-qPCR、蛋白质印迹法、CCK8实验和平板克隆形成实验证实干扰KGA后,肿瘤细胞增殖能力显著降低,GIN-1处理HuH-6细胞后蛋白质印迹法、CCK8实验和平板克隆形成实验证实抑制KGA后肿瘤细胞生长能力降低。结论与正常肝脏相比,KGA在肝母细胞瘤中呈高表达,且高表达的KGA与肝母细胞瘤不良预后相关,是HB患儿预后的独立预测因子,抑制KGA后肝母细胞瘤细胞增殖能力降低。

Objective To explore the expression of kidney-type glutaminase(KGA)in hepatoblastoma(HB),to detect its prognosis correlation and to elucidate the effects of kidney-type glutaminase on the proliferation of HuH-6 cells in vitro.Methods Gene expression profiles and clinical data of HB datasets were retrieved from the database of GEO.Differential gene expression analysis revealed the expression of KGA in normal liver tissues and hepatoblastoma tissues.The pediatric patients were divided into the high expression and the low expression groups based on the median mRNA relative level of KGA(median level=37.181).The survival differences were evaluated by Kaplan-Meier method,and Cox regression analysis was utilized for developing a KGA-related prognostic signature.Using quantitative real-time polymerase chain reaction(RT-qPCR)and western blot,we detected the expression of KGA in hepatoblastoma cell lines and verified the silencing effects of KGA by treating HB cells with siRNA and KGA inhibitor(glutaminase-IN-1,GIN-1).The role of KGA in the proliferation was determined by CCK-8 assay and plate clone formation experiment.Results AS compared with normal liver tissues,the expression of KGA significantly increased in HB tissues.And using Kaplan-Meier survival analysis,children with high KGA expression had a shorter progression-free survival time(P=0.014),Multivariate Cox regression analysis confirmed that KGA was an independent predictor of prognosis in HB patients[HR(95%CI):5.74(1.21,27.33),P=0.028].According to RT-qPCR,Western blotting,CCK8 assay,and plate clone formation assay,the proliferation of HuH-6 cells was suppressed by treating with small interfering RNA or KGA inhibitor.Conclusions KGA may serve as a promising prognostic biomarker and a novel therapeutic target for HB.