葡萄糖调节蛋白78对肝癌预后及肿瘤细胞增殖的影响  

The influence of glucose regulatory protein 78 on prognosis and tumor cell proliferation of hepatocellular carcinoma

作　　者：马海东 曹洁[1] 高龙 付文康 米宁宁 白明圳 林延延[1] 苏刚[2] 寇温[1] 孟文勃[1] Ma Haidong;Cao Jie;Gao Long;Fu Wenkang;Mi Ningning;Bai Mingzhen;Lin Yanyan;Su Gang;Kou Wen;Meng Wenbo(Department of General Surgery,the First Hospital of Lanzhou University,Lanzhou 730013,China;School of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]兰州大学第一医院普通外科,730030 [2]兰州大学基础医学院,730000

出　　处：《中华消化外科杂志》2021年第12期1294-1305,共12页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金(81872036,82060551);2020年甘肃省卫生健康行业科研计划项目(GSWSKY2020-11);甘肃省科技厅青年基金(20JR5RA353)。

摘　　要：目的分析葡萄糖调节蛋白78(glucose regulatory protein 78, GRP78)对肝癌预后及肿瘤细胞增殖的影响。方法采用实验研究方法和回顾性队列研究方法。采用肝癌组织芯片, 体外培养Huh7、Hep3B肝癌细胞和LO2正常肝细胞, 结合免疫组织化学染色、细胞转染、实时荧光定量聚合酶链式反应(qRT-PCR)、Western blot检测、细胞增殖实验、细胞克隆形成实验及高通量转录组学检测分析肝癌细胞GRP78表达情况。Hep3B、Huh7细胞转染GRP78基因特异性shRNA慢病毒设为GRP78-shRNA组, 转染阴性对照shRNA慢病毒设为对照-shRNA组。观察指标:(1)肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系。(2)肝癌病人预后及影响因素分析。(3)抑制GRP78表达对肝癌细胞增殖的影响。(4)抑制GRP78表达对肝癌细胞p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。(5)HA15对肝癌细胞增殖和p53、p21、CDK2、CDK4、CDK6基因和蛋白表达的影响。正态分布的计量资料以x±s表示, 组间比较采用t检验或方差分析。重复测量数据采用重复测量方差分析。计数资料以绝对数表示, 组间比较采用χ2检验。单因素和多因素分析采用COX比例风险回归模型。采用Kaplan-Meier法计算生存时间并绘制生存曲线, 采用Log-rank检验进行生存分析。结果 (1)肝癌组织和癌旁组织GRP78表达及其与临床病理特征的关系:肝癌组织芯片免疫组织化学染色结果显示, GRP78在90例肝癌组织中低表达53例, 高表达37例, GRP78在90例癌旁组织中低表达84例, 高表达6例, 肝癌组织与癌旁组织比较, 差异有统计学意义(P<0.05)。(2)肝癌病人预后及影响因素分析:90例病人均获得随访, 随访时间为5~56个月, 中位随访时间为49个月。53例GRP78低表达肝癌病人中位总体生存时间和中位疾病无进展生存时间分别为56个月和53个月, 37例GRP78高表达肝癌病人上述指标分别为32个月和19个月, 两者比Objective To investigate the influence of glucose regulatory protein 78(GRP78)on prognosis and tumor cell proliferation of hepatocellular carcinoma.Methods The experimental study and retrospective cohort study were conducted.Based on hepatocellular carcinoma tissue chip,in vitro culture of Huh7 and Hep3B hepatoma cells and L02 normal hepatic cell,and combined with immunohistochemical staining,cell transfection,quantitative real-time polymerase chain reaction(qRT-PCR),Western blot detection,cell proliferation experiments,cell clone formation experiments and high-throughput transcription histological analysis,the GRP78 expression in hepatoma cells was analyzed.Huh7 and Hep3B hepatoma cells being transfected with the GRP78 gene-specific shRNA lentiviruses or the negative control shRNA lentivirus were set as the GRP78 gene-specific shRNA lentivirus group and the negative control shRNA lentivirus group respectively.Observation indicators:(1)GRP78 expression in hepatocellular carcinoma tissue and adjacent tissue and its correlation with the clinicopathological characteristics of hepatocellular carcinoma patients;(2)analysis of factors affecting the prognosis of hepatocellular carcinoma patients;(3)effects of inhibiting of GRP78 expression on the proliferation of hepatoma cells;(4)effects of inhibiting of GRP78 expression on the gene and protein expression of p53,p21,CDK2,CDK4,and CDK6 in hepatoma cells;(5)effects of HA15 on the proliferation and the gene and protein expression of p53,p21,CDK2,CDK4,and CDK6 in hepatoma cells.Measurement data of the normal distribution were expressed as MeaA?±5D,and comparison of groups was conducted using the t test or ANOVA.Repeated measurement data were analyzed using repeated ANOVA.Count data were expressed as absolute numbers,and comparisons between groups was conducted using the chi-square test.COX proportional hazards regression model was used for univariate and multivariate analysis.The Kaplan-Meier method was used to calculate the survival time and draw survival curve,and the L

关 键 词：肝肿瘤 葡萄糖调节蛋白78 细胞增殖 靶向抑制剂 预后 

分 类 号：R735.7[医药卫生—肿瘤]