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

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细胞的条件培养基培养HUVEC细胞形成的血管主结点数、主分段数和网格总面积分别为(295.3±26.4)个、(552.5±63.8)个和(2239781.0±138211.9)平方像素,均高于对照组[分别为(113.3±23.5)个、(194.8±36.5)个和(660621.0±280328.3)平方像素,均P<0.01];用LDLR敲降组HLE细胞的条件培养基培养HUVEC细胞形成的血管主结点数、主分段数和网格总面积分别为(245.3±42.4)个、(257.5±20.4)个和(2535754.5±249094.2)平方像素,均高于对照组[分别为(113.3±23.5)个、(114.3±12.2)个和(1565456.5±219259.7)平方像素,均P<0.01];在对照组MHCC-97H细胞条件培养基中加入CEA培养HUVEC细胞形成的血管主结点数、主分段数和网格总面积分别为(178.9±12.0)个、(286.9±12.3)个和(1966990.0±126249.5)平方像素,均高于对照组[分别为(119.7±22.1)个、(202.7±33.7)个和(1421191.0±189837.8)平方像素,均P<0.01]。肝癌组织中LDLR的表达与CEA的表达无相关性,与肝癌组织中CD31的表达、血清CEA水平、血清ALT水平均呈负相关(r=-0.167,P=0.044;r=-0.061,P=0.032;r=-0.147,P=0.05)。肝癌组织中CEA的表达与CD31的表达呈正相关(r=0.192,P=0.020),血清CEA水平与血清ALT水平呈正相关(r=0.164,P=0.029)。结论肝癌细胞敲降LDLR可通过释放CEA促进肝癌血管异常化。

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-regulated and 515 genes were down-regulated.The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group.The Gene Ontology(GO)function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function.The Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch,the extracellular matrix receptor interactions,and the interactions with the extracellular matrix receptors.The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43,which was higher than that of the control group(1.15±0.14,P<0.001).The results of angiogenesis experiments showed that at 5 h,the number of main junctions,the number of main segments,and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4,552.5±63.8,and 2239781.0±138211.9 square pixels,which were higher than those of the control group(113.3±23.5,194.8±36.5,and 660621.0±280328.3 square pixels,respectively,all P<0.01).The number of vascular major junctions,the number of major segments,and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4,257.5±20.4,and 2535754.5±249094.2 square pixels,respectively,which were all higher than those of the control group(113.3±23.5,114.3±12.2,and 1565456.5±219259.7 square pixels,respectively,all P<0.01).In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions,the number of main segments,and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0,286.9±12.3,and 1966990.0±126249.5 spixels,which were higher than those in the control group(119.7±22.1,202.7±33.7,and 1421191.0±189837.8 square pixels,respectively).The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA,but was negatively correlated with the expression of CD31(r=-0.167,P=0.044),the level of serum CEA(r=-0.061,P=0.032),and the level of serum ALT(r=-0.147,P=0.05).The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31(r=0.192,P=0.020).The level of serum CEA was positively correlated with the level of serum ALT(r=0.164,P=0.029).Conclusion Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.