ASGR1在肝细胞癌中的意义及机制研究
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Study on the significance and mechanism of ASGR1 in hepatocellular carcinoma
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通讯作者: 刘艳丰,电子信箱:lyf7858188@163.com。
编委: 徐敏
收稿日期: 2023-08-16 接受日期: 2023-09-18 网络出版日期: 2023-09-28
基金资助: |
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Corresponding authors: LIU Yanfeng, E-mail:lyf7858188@163.com.
Received: 2023-08-16 Accepted: 2023-09-18 Online: 2023-09-28
目的·探究去唾液酸糖蛋白受体1(asialoglycoprotein receptor 1,ASGR1)在肝细胞癌(hepatocellular carcinoma,HCC)中的意义及潜在机制。方法·通过R语言分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中ASGR1在肝癌患者中的表达情况并绘制相关生存曲线。利用人类蛋白质图谱(The Human Protein Atlas,HPA)数据库获得人体正常肝组织和肝癌组织的免疫组织化学(immunohistochemistry,IHC)数据来分析ASGR1的蛋白表达情况。利用流体动力学尾静脉注射(hydrodynamic tail vein injection,HTVI)递送方法,在免疫完全的小鼠肝脏中敲除Asgr1探究其在体内的致瘤功能,并通过蛋白免疫印迹法(Western blotting,WB)验证基因敲除效率。利用R语言进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析及相关性分析,利用基因探针富集(Gene Set Enrichment Analysis,GSEA)软件进行GSEA hallmark相关通路分析,利用实时荧光定量PCR(quantitative real-time PCR,qPCR)在小鼠肝癌组织中验证糖酵解关键基因表达水平。结果·ASGR1在肝癌组织中显著低表达,在肝癌患者中ASGR1的低表达与患者较差的总体生存期(overall survival,OS)、无疾病间隔(disease free interval,DFI)、无进展间隔期(progression free interval,PFI)和疾病特异性生存期(disease specific survival,DSS)相关;肿瘤分级程度越高的肝癌患者ASGR1基因表达水平越低。人体正常肝组织ASGR1蛋白的表达显著高于肝癌组织。在免疫完全的肝细胞癌小鼠模型中,小鼠内源性Asgr1敲除可增加肝组织中肿瘤结节的大小和数量。TCGA数据库中ASGR1低表达组肝癌患者富集到多条癌症及代谢相关通路,ASGR1表达与部分糖酵解关键基因表达呈负相关,Asgr1敲除组的小鼠肝癌组织中糖酵解水平高于对照组,提示ASGR1低表达很可能促进肝癌的生长发展,加强代谢重编程促进肿瘤的合成代谢发展。结论·ASGR1在肝癌患者中表达显著降低,与患者的预后呈正相关;小鼠体内敲除Asgr1可促进肝细胞癌的发生发展;ASGR1可以作为肝癌预后不良的潜在生物标志物和潜在治疗新靶点。
关键词:
Objective ·To explore the significance and mechanism of asialoglycoprotein receptor 1 (ASGR1) in hepatocellular carcinoma. Methods ·The expression of ASGR1 in patients with liver cancer in The Cancer Genome Atlas (TCGA) database was analyzed by R language and the related survival curves were drawn. The Human Protein Atlas (HPA) database was used to obtain the immunohistochemistry (IHC) data of normal human liver tissue and liver cancer tissue to analyze the protein expression of ASGR1. By using the hydrodynamic tail vein injection (HTVI) delivery method, Asgr1 was knocked out in the liver of fully immune mice to explore its tumorigenic function invivo. Gene knockout efficiency was verified by Western blotting (WB). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and correlation analysis were performed by using R language. The GSEA hallmark correlation pathway analysis was performed by using Gene Set Enrichment Analysis (GSEA) software. The expression level of key genes of glycolysis in mouse liver cancer tissue was verified by quantitative real-time PCR (qPCR). Results ·ASGR1 was significantly low-expressed in liver cancer tissue, and the low expression of ASGR1 in liver cancer patients was associated with poorer overall survival (OS), disease-free interval (DFI), progression-free interval (PFI), and disease-specific survival (DSS). The higher the degree of tumor grade, the lower the expression level of ASGR1 in patients with liver cancer. Immunohistochemistry showed that the protein expression of ASGR1 in normal human liver tissue was significantly higher than that in liver cancer tissue. In an immunocompetent mouse model of hepatocellular carcinoma, knockout of endogenous Asgr1 in mice increased the size and number of tumor nodules in liver tissue. In the TCGA database, patients with liver cancer in the ASGR1 low-expression group were enriched in multiple cancer and metabolic pathways. The expression of ASGR1 was negatively correlated with some key genes of glycolysis. The level of glycolysis in liver cancer tissues of mice in the Asgr1 knockout group was higher than that in the control group. It was suggested that the low expression of ASGR1 be likely to promote the growth and development of liver cancer and strengthen metabolic reprogramming to promote the anabolic development of tumors. Conclusion ·The expression of ASGR1 is significantly reduced in patients with liver cancer, which is positively correlated with the prognosis of patients. Knocking out Asgr1 in mice can promote the occurrence and development of hepatocellular carcinoma. ASGR1 can be used as a potential biomarker for poor prognosis of liver cancer and a new target for potential treatment.
Keywords:
本文引用格式
李倩玉, 郭文韵, 钱逸斐, 李松玲, 朱子俊, 刘艳丰.
LI Qianyu, GUO Wenyun, QIAN Yifei, LI Songling, ZHU Zijun, LIU Yanfeng.
肝癌(liver cancer)是世界上最常见的癌症之一,原发性肝癌(primary liver cancer)在全球癌症死因中位于第3位,严重威胁着人类健康[1-2]。原发性肝癌有多种类型,其中肝细胞癌(hepatocellular carcinoma,HCC)占全球肝癌病例的75%~85%,是最常见的一种肝癌类型。肝细胞癌的发生发展是一个多步骤的病理过程,发病的危险因素也多种多样,如乙型肝炎病毒和丙型肝炎病毒的慢性感染、黄曲霉毒素的感染、长期饮酒、2型糖尿病等[3-4]。虽然目前对肝细胞癌的研究已取得一定进展,但是其发生和发展机制尚未清晰,了解肝细胞癌的潜在分子机制对于未来的治疗至关重要。
1 材料与方法
1.1 数据收集
通过UCSC Xena(xenabrowser.net)网站获取癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库的基因表达矩阵和患者临床数据。人体正常肝组织及肝癌组织相关的免疫组织化学(immunohistochemistry,IHC)数据来自人类蛋白质图谱(The Human Protein Atlas,HPA)数据库(
1.2 生物信息学分析
使用R语言对基因表达矩阵进行癌和癌旁组织的表达量分析及肝癌患者不同基因表达之间的相关性分析。使用R语言survival包和survminer包进行生存分析。使用R语言limma包对基因表达矩阵按照ASGR1的中位值分为高低2组后进行差异基因分析,通过P值及错误发现率(false discovery rate,FDR)筛选差异基因,使用R语言ClusterProfiler包进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,使用基因探针富集(Gene Set Enrichment Analysis,GSEA)软件进行基因集富集分析。
1.3 流体动力学尾静脉注射
使用CRISPR-Cas9方法构建小鼠Asgr1-sgRNA质粒。将6周大的雄性C57BL/6小鼠随机分组,每组5只小鼠,利用流体动力学尾静脉注射(hydrodynamic tail vein injection,HTVI)方法,每只小鼠注射2 mL溶于生理盐水的质粒混合物。每2 mL质粒混合物包含12 μg pT3-EF1A-MYC-IRES-luciferase、10 μg pX330-sg-p53和6.5 μg CMV-SB13。试验组或对照组再分别加入12 μg lenti-CRISPR sgAsgr1或12 μg空载体lenti-CRISPR v2。在5~7 s内将溶于生理盐水的质粒混合物注射到小鼠的尾静脉中。通过腹部触诊监测小鼠,并在它们具有高肝肿瘤负荷时安乐死,取肝脏肿瘤进行分析。构建Asgr1-sgRNA质粒的引物序列见表1。
表1 sgRNA序列(5′→3′)
Tab 1
sgRNA | Forward (5′→3′) | Reverse (5′→3′) |
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Asgr1-sgRNA-1 | CACCGCGCTGCAAGAGCCGTGGAGT | AAACACTCCACGGCTCTTGCAGCGC |
Asgr1-sgRNA-2 | CACCGGACCATCATCAACTCCGGAG | AAACCTCCGGAGTTGATGATGGTCC |
1.4 蛋白免疫印迹法
液氮研磨小鼠肿瘤组织后,使用RIPA蛋白裂解液冰上裂解30 min,离心获得蛋白上清液,用酶标仪进行蛋白定量,后与蛋白上样缓冲液一起煮沸,获得蛋白样品。取适量蛋白样品上样后进行蛋白凝胶电泳,后将蛋白质转膜到PVDF膜上。用5%脱脂牛奶室温封闭1 h,洗膜后一抗4 ℃孵育过夜。次日洗膜后室温下孵育二抗1 h,洗膜后在凝胶成像仪中成像并记录。
抗体信息:ASGR1抗体(PTM BIO,PTM-6093),ACTB抗体(ABclonal,AC028)。
1.5 总RNA提取及实时荧光定量PCR
液氮研磨小鼠肿瘤组织后,加入适量TRIzol进行裂解,后加入体积为TRIzol体积1/5的三氯甲烷颠倒混匀。12 000×g低温离心15 min后收集上清液,加入等体积异丙醇后混匀。继续12 000×g低温离心10 min,弃上清液并加入75%乙醇。12 000×g低温离心5 min后弃上清液。移除乙醇并自然干燥RNA沉淀,及时溶解于DEPC水并测定RNA浓度。使用反转录试剂将RNA反转录为cDNA。后在罗氏LightCycler®384系统进行实时荧光定量PCR(quantitative real-time PCR,qPCR)反应。引物序列见表2。
表2 PCR引物序列(5′→3′)
Tab 2
Primer | Forward (5′→3′) | Reverse (5′→3′) |
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Actb | GGCTGTATTCCCCTCCATCG | CCAGTTGGTAACAATGCCATGT |
Hk2 | TGATCGCCTGCTTATTCACGG | AACCGCCTAGAAATCTCCAGA |
Pgk1 | ATGTCGCTTTCCAACAAGCTG | GCTCCATTGTCCAAGCAGAAT |
Pkm2 | GCCGCCTGGACATTGACTC | CCATGAGAGAAATTCAGCCGAG |
1.6 统计学方法
采用R软件进行生物信息学分析。试验数据使用GraphPad Prism 9软件进行分析,组间比较采用t检验,P<0.05表示差异具有统计学意义。
2 结果
2.1 ASGR1 在肝细胞癌中的表达及其与预后的关系
为了探究ASGR1与肝细胞癌发生发展的关系,我们在TCGA肝癌数据库中对临床患者的转录组测序数据进行生物信息学分析,结果显示:相比于癌旁组织,ASGR1在肝癌组织样本中低表达(图1A);随着肝癌进展,分级越高,ASGR1的表达越低(图1B)。根据TCGA数据库肝癌患者的临床信息绘制生存曲线,结果显示ASGR1的表达与肝癌患者的总体生存期(overall survival,OS)、无疾病间隔(disease-free interval,DFI)、无进展间隔期(progression-free interval,PFI)和疾病特异性生存期(disease-specific survival,DSS)均呈明显的正相关关系(图1C~F)。
图1
图1
ASGR1 在肝癌组织样本中的表达及其与预后的关系
Note: A. Expression of ASGR1 in tumor and normal tissues of liver cancer patients from TCGA database. B. Expression of ASGR1 in different stages of liver cancer patients from TCGA database. C‒F. The overall survival (C), disease-free interval (D), progression-free interval (E) and disease-specific survival (F) curves of liver cancer patients with low or high ASGR1 expression from TCGA database.
Fig 1
Expression of ASGR1 in liver cancer samples and its correlation with prognosis
2.2 ASGR1蛋白在肝癌组织中的表达情况
通过HPA数据库,我们获得了人体正常肝组织和肝癌组织相关的IHC数据,以比较两者之间ASGR1的蛋白表达水平。结果显示,ASGR1在肝癌组织中的表达显著低于正常肝组织(图2A、B)。
图2
图2
ASGR1在人体正常肝组织和肝癌组织的蛋白表达水平
Note: A. Immunohistochemical pictures of ASGR1-related protein expression levels in normal human liver tissue and liver cancer tissue from the HPA database. B. The proportion of different degrees of ASGR1 staining in normal human liver tissue and liver cancer tissue.
Fig 2
Protein expression level of ASGR1 in normal human liver tissue and liver cancer tissue
2.3 Asgr1 敲除在小鼠体内对肝细胞癌发生发展的影响
图3
图3
小鼠流体动力学高压尾静脉注射模型
Note: Delivery via HTVI in fully immune mice by injection of pT3-EF1A-MYC-IRES-luciferase, pX330-sg-p53, CMV-SB13, lenti-CRISPR sgAsgr1 (sgAsgr1 group) or lenti-CRISPR v2 (sgCtrl group).
Fig 3
Schematic diagram of mouse hydrodynamic tail vein injection model
图4
图4
Asgr1 敲除验证及小鼠肝细胞癌的形成情况
Note: A. Representative pictures of hepatocellular carcinoma in sgCtrl and sgAsgr1 group. B. Protein expression of ASGR1 and ACTB in liver tumor tissues of mice in sgCtrl and sgAsgr1 group. C‒F. Liver weight ratio (C), tumor number (D), max size (E) and proportion of tumor numbers (F) with different sizes in sgCtrl and sgAsgr1 group.
Fig 4
Validation of Asgr1 knockout and formation of hepatocellular carcinoma in mice
2.4 ASGR1 相关的生物学功能及富集通路分析
图5
图5
TCGA肝癌数据库中 ASGR1 表达相关的生物信息学分析及小鼠肝癌组织mRNA表达水平验证
Note: A. KEGG pathway enrichment analysis of genes negatively correlated with ASGR1. B. GSEA analysis of hallmark pathway enrichment between ASGR1 low and high group. C. Correlation analysis of expression of ASGR1 and glycolysis-related genes. D. The mRNA expression levels of glycolysis-related genes in mouse liver cancer tissue.
Fig 5
Bioinformatics analysis of ASGR1 expression in TCGA liver cancer database and verification of mRNA expression levels in mouse liver cancer tissue
3 讨论
ASGR1,也被称为肝细胞半乳糖/N-乙酰基葡糖胺受体,或者Ashwell-Morell受体,主要表达于肝窦状间隙的肝实质细胞表面[14],能够介导去唾液酸化的糖蛋白被肝细胞内吞降解[15]。目前已知的ASGR1在糖蛋白代谢[16]、调节凝血[17]、胆固醇代谢[18]等多个生理过程都发挥着重要的作用[19]。在肝病中,发现ASGR1在肝硬化组织中上调[20]。关于ASGR1在肝细胞癌中的作用,有研究[21]表明,ASGR1可能通过与LASS2相互作用来抑制V-ATPase的活性,从而抑制肝癌细胞的转移潜能。最新的研究[22]表明,ASGR1通过促进NLK与STAT3的结合、抑制STAT3磷酸化来抑制肝癌的进展。这些研究均提示ASGR1在肝细胞癌发生发展中发挥着重要的作用,然而关于ASGR1在肝细胞癌中的研究仍然很少。
本研究为了探究ASGR1在肝细胞癌中的意义及机制,首先分析TCGA数据库中ASGR1在肝癌患者中的表达情况并做了生存分析,发现ASGR1在肝癌组织中显著低表达,同时肿瘤分级程度越高,ASGR1基因表达水平越低;且ASGR1低表达的患者的OS、DFI、PFI、DSS均缩短。之后,又利用HPA数据库发现人体正常肝组织ASGR1蛋白的表达显著高于肝癌组织。以上结果说明,ASGR1基因很可能是一种肿瘤抑制因子,在肝细胞癌进展中发挥抗肿瘤作用。为了更有效地探究ASGR1在肝细胞癌发生发展中的作用,通过HTVI建立小鼠肝原位模型,在小鼠肝脏中敲除Asgr1,探究其在体内的致瘤的功能,结果表明,小鼠内源性敲除Asgr1可以增加肝组织中肿瘤结节的大小和数量,敲除Asgr1后肝细胞癌体内成瘤能力增强。通过对TCGA中肝癌的数据进行KEGG及GSEA分析,发现ASGR1的低表达激活了癌症相关通路以及细胞周期和细胞增殖等相关通路,提示ASGR1的低表达有助于肝细胞癌的生长和发展;同时,ASGR1低表达组与糖酵解通路、PI3K-AKT-MTOR信号通路有关,相关性分析表明ASGR1与糖酵解通路部分关键基因的表达水平呈显著的负相关。为了进一步证实分析的结果,我们在小鼠肝癌组织中对糖酵解通路部分关键基因在mRNA水平进行验证,验证结果与生物信息学分析一致,Asgr1敲除组小鼠肝癌组织的糖酵解水平高于对照组。肝脏作为代谢器官在维持机体代谢稳态中起着重要作用,在调节各种代谢途径方面具有重要作用[23],代谢重编程被认为是肿瘤重要的代谢表型[24-25],恶性肿瘤可以通过改变代谢途径来维持生长复制需要的高能量需求[26]。参与糖酵解、脂肪生成、氨基酸合成和核苷酸生物合成的几种代谢酶得到增强[27],以加强代谢重编程过程中肿瘤的合成代谢发展。致癌信号转导通路PI3K-AKT-MTOR通路可增强肿瘤中的Warburg效应,促进癌细胞生长和转移[28];该信号激活的癌症已被发现变得更具侵袭性,并且AKT通路激活已被证明是肝癌患者早期复发和预后不良的显著危险因素[29-30]。这些均提示ASGR1的低表达很可能促使肝细胞癌发生代谢重编程,进而促进肝细胞癌的发生发展。其具体机制还有待进一步探究。
综上所述,本研究主要通过生物信息学分析和小鼠体内尾静脉注射模型探究了ASGR1在肝细胞癌中的意义及机制,提出ASGR1基因具有作为肝细胞癌临床预后指标和治疗靶点的潜力,并初步分析验证了ASGR1低表达促进肝细胞癌发生发展的分子机制及信号通路。本研究可为肝细胞癌发生发展及治疗提供新的思路。
作者贡献声明
李倩玉、郭文韵参与试验设计;李倩玉、钱逸斐负责论文的写作和修改;李松玲、朱子俊参与文献检索;刘艳丰负责试验指导与论文审阅。所有作者均阅读并同意最终稿件的提交。
AUTHOR's CONTRIBUTIONS
The study was designed by LI Qianyu and GUO Wenyun. The manuscript was drafted and revised by LI Qianyu and QIAN Yifei. The document was searched by LI Songling and ZHU Zijun. The research was guided by LIU Yanfeng. All the authors have read the last version of paper and consented for submission.
利益冲突声明
所有作者声明不存在利益冲突。
COMPETING INTERESTS
All authors disclose no relevant conflict of interests.
参考文献
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