LMO7通过靶向铁死亡促进肝细胞癌生长

LMO7 promotes the growth of hepatocellular carcinoma by targeting ferroptosis

目的 探讨LMO7在肝细胞癌(肝癌)中的表达及其调控肝癌增殖和生长的潜在作用机制。方法 基于Kaplan-Meier Plotter数据库进行生物信息学分析,验证LMO7与肝癌患者预后的关系。使用shRNA病毒感染后的肝癌细胞HepG2和PLC/PRF/5分成空白对照组(NC)、LMO7敲低组1(shLMO7#1)、LMO7敲低组2(shLMO7#2)3组。采用Western blot和qRT-PCR验证肝癌细胞中LMO7蛋白和mRNA相对表达量,细胞克隆形成实验检测LMO7表达对HepG2细胞增殖能力的影响。采用Western blot检测敲低LMO7后铁死亡相关蛋白ACSL4和P53的表达;流式细胞术检测敲低LMO7后的细胞周期和活性氧(ROS)表达量。两组LMO7表达比较采用t检验,3组比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果 基于GEO数据库分析显示,肝癌组织LMO7表达明显低于癌旁组织(LSD-t=-3.038,P<0.05)。基于Kaplan-Meier Plotter数据库分析显示,肝癌组织LMO7低表达与患者的不良预后相关(HR=0.50,P<0.05)。细胞克隆形成实验显示,shLMO7#1组和shLMO7#2组平均细胞克隆数分别为(64±10)、(95±26)个,明显多于NC组的(11±5)个(LSD-t=3.91,6.27;P<0.05)。Western blot检测显示,下调LMO7后肝癌细胞P53蛋白表达显著下调。流式细胞术检测显示,下调LMO7后HepG2细胞主要阻滞在G2/M期;shLMO7#1组和shLMO7#2组LMO7的ROS相对表达量分别为32.6±1.6、47.9±1.0,明显少于NC组的53.3±1.1(LSD-t=-20.12,-5.27;P<0.05)。结论 LMO7在肝癌患者中低表达,LMO7低表达与患者预后不良有关。低表达LMO7通过靶向抑制铁死亡相关基因P53的表达,进一步调控ROS和细胞周期,从而促进肝癌生长。

Objective To investigate the expression of LMO7 in hepatocellular carcinoma (HCC) and the potential mechanism of regulating the proliferation and growth of HCC.Methods Bioinformatics analysis was carried out based on Kaplan-Meier Plotter database to validate the correlation between LMO7 and prognosis of HCC patients.HepG2 and PLC/PRF/5 HCC cells infected with shRNA virus were divided into 3 groups:normal control group (NC),LMO7 knockdown group 1 (shLMO7#1) and LMO7 knockdown group 2 (SHLMO7#2).The relative expression levels of LMO7 protein and mRNA in HCC cells were determined by Western blot and qRT-PCR.The effect of LMO7 expression on the proliferation of HepG2 cells was assessed by cell colony formation assay.The expression levels of ferroptosis-related proteins ACSL4 and P53 after LMO7 knockdown were detected by Western blot.The cell cycle and expression level of reactive oxygen species (ROS) were assessed by flow cytometry.The expression levels of LMO7 between two groups were compared by t test.The comparison among three groups was performed by one-way ANOVA.Paired comparison between two groups was conducted by LSD-t test.Results GEO database analysis showed that the expression of LMO7 in HCC was significantly lower than that in paracancerous tissues (LSD-t=-3.038,P<0.05).Kaplan-Meier Plotter database analysis showed that low expression of LMO7 in HCC tissues was significantly correlated with poor prognosis of HCC patients (HR=0.50,P<0.05).Cell colony formation assay found that the average number of cell colony in the shLMO7#1 and shLMO7#2 groups was 64±10 and 95±26,significantly higher than 11±5 in the NC group (LSD-t=3.91,6.27;P<0.05).Western blot showed that the expression level of P53 protein in HCC cells was significantly down-regulated after knockdown of LMO7.Flow cytometry showed that HepG2 cells were mainly arrested in G2/Mphase after down-regulation of LMO7.The relative ROS expression levels of LMO7 in the shLMO7#1 and shLMO7#2 groups were 32.6±1.6 and 47.9±1.0,significantly lower than 53.3±1.1 in the NC group (LSD-t=-20.12,-5.27;P<0.05).Conclusions LMO7 is lowly expressed in HCC patients,which is associated with poor prognosis of HCC patients.Low expression of LMO7 further regulates ROS and cell cycle through targeted inhibition of ferroptosis-related gene P53,thereby promoting the growth of HCC.