ASGR1在肝细胞癌中的意义及机制研究

doi:10.3969/j.issn.1674-8115.2023.09.005

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (9): 1107-1114.doi: 10.3969/j.issn.1674-8115.2023.09.005

• 论著 · 基础研究 • 上一篇

ASGR1在肝细胞癌中的意义及机制研究

李倩玉¹(), 郭文韵¹, 钱逸斐¹, 李松玲², 朱子俊¹, 刘艳丰¹^,³()

^1.上海交通大学医学院附属仁济医院临床干细胞研究中心，上海 200127
^2.上海交通大学生物医学工程学院Med-X研究院，上海 200030
^3.上海交通大学医学院附属仁济医院肝外科，上海 200127

收稿日期:2023-08-16 接受日期:2023-09-18 出版日期:2023-09-28 发布日期:2023-09-28
通讯作者: 刘艳丰 E-mail:qianyu_lli@163.com;lyf7858188@163.com
作者简介:李倩玉（1997—），女，硕士生；电子信箱：qianyu_lli@163.com。
基金资助:
国家自然科学基金(82073190);上海交通大学医学院“双百人”项目(20221704)

Study on the significance and mechanism of ASGR1 in hepatocellular carcinoma

LI Qianyu¹(), GUO Wenyun¹, QIAN Yifei¹, LI Songling², ZHU Zijun¹, LIU Yanfeng¹^,³()

^1.Renji-Med X Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.School of Biomedical Engineering & Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China
^3.Department of Liver Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2023-08-16 Accepted:2023-09-18 Online:2023-09-28 Published:2023-09-28
Contact: LIU Yanfeng E-mail:qianyu_lli@163.com;lyf7858188@163.com
Supported by:
National Natural Science Foundation of China(82073190);“Two-hundred Talents”Program of Shanghai Jiao Tong University School of Medicine(20221704)

摘要/Abstract

摘要：

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: 去唾液酸糖蛋白受体1（ASGR1）, 肝细胞癌, 流体动力学尾静脉注射, 治疗靶点

Abstract:

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.

Key words: asialoglycoprotein receptor 1 (ASGR1), hepatocellular carcinoma, hydrodynamic tail vein injection, therapeutic target

中图分类号:

R735.7

李倩玉, 郭文韵, 钱逸斐, 李松玲, 朱子俊, 刘艳丰. ASGR1在肝细胞癌中的意义及机制研究[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1107-1114.

LI Qianyu, GUO Wenyun, QIAN Yifei, LI Songling, ZHU Zijun, LIU Yanfeng. Study on the significance and mechanism of ASGR1 in hepatocellular carcinoma[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1107-1114.

图/表 7

表1 sgRNA序列（5′→3′）

Tab 1 Sequence for sgRNA （5′→3′）

sgRNA	Forward (5′→3′)	Reverse (5′→3′)
Asgr1-sgRNA-1	CACCGCGCTGCAAGAGCCGTGGAGT	AAACACTCCACGGCTCTTGCAGCGC
Asgr1-sgRNA-2	CACCGGACCATCATCAACTCCGGAG	AAACCTCCGGAGTTGATGATGGTCC

表2 PCR引物序列（5′→3′）

Tab 2 Primer sequence for PCR （5′→3′）

Primer	Forward (5′→3′)	Reverse (5′→3′)
Actb	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
Hk2	TGATCGCCTGCTTATTCACGG	AACCGCCTAGAAATCTCCAGA
Pgk1	ATGTCGCTTTCCAACAAGCTG	GCTCCATTGTCCAAGCAGAAT
Pkm2	GCCGCCTGGACATTGACTC	CCATGAGAGAAATTCAGCCGAG

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

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

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 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

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

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ASGR1在肝细胞癌中的意义及机制研究

Study on the significance and mechanism of ASGR1 in hepatocellular carcinoma

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