敲降低密度脂蛋白受体相关蛋白表达对肝癌血管异常化的影响及其机制  

The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms

作　　者：武强 詹琳琳 王宇 贺钰超 陈璐 陈子叶 李广涛 刘东明 包旭 刘潇濛 郭华 宋天强 Wu Qiang;Zhan Linlin;Wang Yu;He Yuchao;Chen Lu;Chen Ziye;Li Guangtao;Liu Dongming;Bao Xu;Liu Xiaomeng;Guo Hua;Song Tianqiang(Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Department of Hepatobiliary Cancer,Tianjin 300060,China;Tianjin Medical University Cancer Institute and Hospital,National Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy,Department of Tumor Cell Biology,Tianjin 300060,China;Beijing Tsinghua Changgung Hospital,Center for Clinical and Translational Science,Beijing 102218,China)

机构地区：[1]天津医科大学肿瘤医院肝胆肿瘤科,国家恶性肿瘤临床医学研究中心,天津市肿瘤防治重点实验室,天津300060 [2]天津医科大学肿瘤医院肿瘤细胞生物学实验室,国家恶性肿瘤临床医学研究中心,天津市肿瘤防治重点实验室,天津300060 [3]北京清华长庚医院临床转化科学中心,北京102218

出　　处：《中华肿瘤杂志》2024年第5期399-408,共10页Chinese Journal of Oncology

摘　　要：目的探讨低密度脂蛋白受体相关蛋白(LDLR)表达对肝癌血管异常化的影响及其机制。方法从Oncomine癌症基因芯片数据库提取87例肝癌组织的基因转录组信息,分析肝癌组织中LDLR的表达水平与癌胚抗原(CEA)和CD31表达水平的相关性。采用短发卡RNA靶基因慢病毒转染的方法,构建LDLR敲降的MHCC-97H和HLE肝癌细胞。通过转录组测序、实时荧光定量聚合酶链反应、蛋白免疫印迹法检测LDLR敲降后肝癌细胞的差异基因及其表达水平变化。通过富集分析明确LDLR参与的基因相关信号通路。通过对肝癌细胞条件培养基中CEA含量的检测评估LDLR对CEA的影响。采用血管生成实验检测LDLR对人脐静脉内皮细胞血管生成能力的影响,以及CEA在LDLR调控血管生成过程中的作用。收集2019年1月至2022年12月于天津医科大学肿瘤医院手术切除的肝癌组织标本176例,采用免疫组织化学染色检测176例肝癌组织中LDLR及146例肝癌组织中CEA和CD31的表达水平,分析肝癌组织中LDLR的表达水平与肝癌组织中CEA和CD31的表达水平、血清CEA和丙氨酸氨基转移酶(ALT)水平的相关性。结果Oncomine数据库分析显示,发生门静脉转移的肝癌患者肝癌组织中LDLR和CEA的表达呈负相关(r=-0.64,P=0.001),而CEA和CD31的表达呈正相关(r=0.46,P=0.010);转录组测序结果显示,LDLR敲降组与对照组MHCC-97H细胞的差异表达基因共有1032个,其中517个基因表达上调,515个基因表达下调。CEA相关细胞黏附分子5(CEACAM5)在LDLR敲降组细胞中上调明显。基因本体论功能富集分析显示,差异基因最明显富集在血管生成的功能上。京都基因与基因组百科全书信号通路富集分析显示,涉及的相关通路主要包括细胞粘着斑、细胞外基质受体相互作用等。LDLR敲降组条件培养基中CEA含量为43.75±8.43,高于对照组(1.15±0.14,P<0.001)。血管生成实验结果显示,在5 h,用LDLR敲降组MHCC-97H细胞�Objectives To investigate the effect of the expression of low-density lipoprotein receptor associated protein(LDLR)on the vascular abnormalities in hepatocellular carcinoma(HCC)and its mechanisms.Methods Based on the information of Oncomine Cancer GeneChip database,we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen(CEA)and CD31 in hepatocellular carcinoma tissues.Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells.The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing,real-time fluorescence quantitative polymerase chain reaction,and protein immunoblotting.The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis.The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells.Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells,as well as the role of CEA in the regulation of angiogenesis by LDLR.Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues,and CEA and CD31 in 146 hepatocellular carcinoma tissues,and analyze the correlations between the expression levels of LDLR,CEA,and CD31 in the tissues,serum CEA,and alanine transaminase(ALT).Results Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated(r=-0.64,P=0.001),whereas the expressions of CEA and CD31 in these tissues were positively correlated(r=0.46,P=0.010).The transcriptome sequencing results showed that there were a total of 1032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells,of which 517 genes were up-regul

关 键 词：肝细胞癌 低密度脂蛋白受体相关蛋白 癌胚抗原 血管生成 

分 类 号：R735.7[医药卫生—肿瘤]