蛋白质组学分析蛋白溶酶体酸性葡糖神经酰胺酶在肝细胞癌中的促增殖作用  

Proteomic analysis of the pro-proliferative role of the protein lysosomal acid glucosylceramidase in hepatocellular carcinoma

作　　者：肖彤 王浩 李红乐 Xiao Tong;Wang Hao;Li Hongle(Department of Clinical Laboratory,the Affiliated Cancer Hospital of Zhengzhou University&Henan Cancer Hospital,Zhengzhou 450008,China;Henan Province Academy of Medical Sciences,Zhengzhou 451162,China)

机构地区：[1]郑州大学附属肿瘤医院(河南省肿瘤医院)检验科,郑州450008 [2]河南省医学科学院,郑州451162

出　　处：《中华肝胆外科杂志》2025年第3期219-225,共7页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金(82203572);河南省医学科技攻关省部共建重大项目(SBGJ20211008)。

摘　　要：目的通过整合分析大规模肝细胞癌临床样本蛋白质组学数据集,挖掘关键促癌分子,并分析其在肝癌细胞中作用机制。方法基于已报道的两个大规模肝细胞癌研究样本蛋白质组学数据集,筛选出癌组织和癌旁组织的差异表达蛋白溶酶体酸性葡糖神经酰胺酶(GBA),癌症基因组图谱(TCGA)数据库分析GBA高表达(n=118)与低表达(n=118)的生存差异。收集2021年3月至2021年5月在河南省肿瘤医院接受手术治疗的8例肝细胞癌患者癌组织和癌旁组织标本,其中男性6例,女性2例,年龄范围41~66岁,中位年龄56岁。人肝癌细胞系HCC-LM3分为转染GBA的小干扰RNA的siGBA-1、siGBA-2组和转染阴性对照的siNC组。Western印迹检测肝癌组织和细胞中相关蛋白的表达。定量蛋白质组学分析肝癌细胞差异表达蛋白,使用Cytoscape(3.9.1)软件对下调差异蛋白进行相互作用网络构建。细胞增殖实验及克隆形成分析GBA表达对肝癌细胞的影响。结果在两个临床样本蛋白质组学数据集中筛选出GBA在肝癌组织中显著上调,TCGA分析GBA高表达与肝细胞癌预后不良相关。Western印迹检测临床肝细胞癌组织中GBA蛋白相对表达为(0.60±0.35),高于癌旁组织(0.26±0.20),差异有统计学意义(t=2.84,P=0.025)。对敲低GBA与阴性对照组细胞进行定量蛋白质组学以及相互作用分析,发现表皮生长因子受体(EGFR)是潜在的GBA下游调控分子。Western印迹检测证实敲低GBA后EGFR蛋白表达显著下降,EGFR蛋白相对表达siNC组为(0.92±0.08)、siGBA-1组为(0.64±0.07)、siGBA-2组为(0.51±0.07)。细胞增殖及克隆形成实验中,敲低GBA后细胞增殖和克隆形成能力均显著下降,细胞增殖第4天细胞吸光度值:siNC组为(1.91±0.17)、siGBA-1组为(1.24±0.11)、siGBA-2组为(1.21±0.04);细胞克隆数:siNC组为(674.3±6.5)、siGBA-1组为(388.3±7.5)、siGBA-2组为(360.0±29.0),敲低组均低于阴性对照组,差异均有统计学意义(均P<0.0Objective To integrate and analyze large-scale proteomic datasets from clinical hepatocellular carcinoma(HCC)samples to identify key oncogenic proteins and investigate their underlying mechanisms.Methods Based on two publicly reported large-scale proteomic datasets of hepatocellular carcinoma,we identified lysosomal acid glucosylceramidase(GBA)as a differentially expressed protein between tumor and adjacent non-tumor tissues.The Cancer Genome Atlas(TCGA)database was utilized to analyze survival differences between patients with high(n=118)and low(n=118)GBA expression.Tumor and adjacent non-tumor tissue samples were collected from eight HCC patients who underwent surgical treatment at Henan Cancer Hospital between March 2021 and May 2021.Among them,six were male and two were female,ranging from 41 to 66 years old,with a median age of 56 years.The human HCC cell line HCC-LM3 was transfected with siRNAs targeting GBA(siGBA-1 and siGBA-2)or a negative control(siNC).Western blot analysis was performed to assess the expression of relevant proteins in HCC tissues and cells.Quantitative proteomic analysis was conducted to identify differentially expressed proteins in HCC cells,and bioinformatics tools were used to construct an interaction network of downregulated proteins.Cell proliferation and colony formation assays were conducted to evaluate the impact of GBA expression on HCC cell growth.Results GBA was identified as a significantly upregulated protein in HCC tissues across two clinical proteomic datasets.TCGA analysis further revealed that high GBA expression was associated with poor prognosis in HCC patients.Western blot analysis confirmed that the relative expression level of GBA protein in clinical HCC tissues was(0.60±0.35),which was significantly higher than that in adjacent non-tumor tissues(0.26±0.20)(t=2.84,P=0.025).Quantitative proteomic analysis interaction analysis of GBA knockdown and control cells identified epidermal growth factor receptor(EGFR)as a potential downstream regulatory target of GBA.Weste

关 键 词：癌 肝细胞 蛋白质组学 细胞增殖 

分 类 号：R735.7[医药卫生—肿瘤]