癌-睾丸抗原SPANXB在肝癌中的表达及其影响肝癌进展的机制研究

doi:10.3969/j.issn.1674-8115.2024.07.001

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (7): 801-813.doi: 10.3969/j.issn.1674-8115.2024.07.001

• 创新团队成果专栏 • 下一篇

癌-睾丸抗原SPANXB在肝癌中的表达及其影响肝癌进展的机制研究

薛煜¹()(), 张海龙²(), 雷鸣¹()

^1.上海交通大学医学院附属第九人民医院上海精准医学研究院，上海 200125
^2.上海交通大学转化医学研究院，上海 200240

收稿日期:2023-12-18 接受日期:2024-04-17 出版日期:2024-07-28 发布日期:2024-07-28
通讯作者: 张海龙,雷鸣 E-mail:yu.xue@sjtu.edu.cn;zhanghailong@sjtu.edu.cn;leim@shsmu.edu.cn
作者简介:薛　煜（1996—），男，硕士生；电子信箱：yu.xue@sjtu.edu.cn。第一联系人：#为共同通信作者。
基金资助:
国家重点研发计划(2018YFA0107004)

Expression of cancer-testis antigen SPANXB and its mechanism in affecting hepatocellular carcinoma progress

XUE Yu¹()(), ZHANG Hailong²(), LEI Ming¹()

^1.Shanghai Institute of Precision Medicine, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^2.Institute of Translational Medicine, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2023-12-18 Accepted:2024-04-17 Online:2024-07-28 Published:2024-07-28
Contact: ZHANG Hailong,LEI Ming E-mail:yu.xue@sjtu.edu.cn;zhanghailong@sjtu.edu.cn;leim@shsmu.edu.cn
Supported by:
National Key Research and Development Program of China(2018YFA0107004)

摘要/Abstract

摘要：

目的·分析癌-睾丸抗原（cancer-testis antigen，CTA）家族成员SPANXB（sperm protein associated with the nucleus on the X chromosome B）在肝癌中的表达及其与肝癌患者预后之间的相关性，并探究SPANXB对肝癌细胞增殖的影响及其潜在机制。方法·利用癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库中的肝癌样本数据，分析SPANXB在肝癌组织中的表达及其与患者生存期的相关性。构建稳定敲低SPANXB与稳定过表达SPANXB的肝癌细胞系，利用活细胞成像实验、EdU细胞增殖实验和平板克隆形成实验评估SPANXB对肝癌细胞增殖的影响。通过RNA测序（RNA-sequence，RNA-seq）探究SPANXB调控肝癌细胞增殖的相关通路，并利用细胞周期实验验证SPANXB对肝癌细胞周期的影响。采用免疫沉淀-质谱联用技术（immunoprecipitation-mass spectrometry，IP-MS）探索与SPANXB相互作用的蛋白，并使用免疫共沉淀（co-immunoprecipitation，Co-IP）进行验证。结果·SPANXB mRNA在肝癌组织中的表达高于正常组织（P=0.003），且与肝癌患者的生存期呈负相关。稳定敲低SPANXB可降低肝癌细胞的增殖能力、克隆形成能力，而稳定过表达SPANXB则可促进这些过程。RNA-seq的结果显示，SPANXB的敲低可下调DNA复制与G1/S细胞周期转换相关通路，细胞周期实验的结果显示SPANXB的敲低可导致肝癌细胞周期发生改变。IP-MS和Co-IP结果显示，SPAXNB与有丝分裂停滞缺陷2样蛋白1（mitotic arrest deficient 2-like protein 1，MAD2L1）、WD重复域蛋白5（WD repeat domain 5，WDR5）等细胞周期相关蛋白存在相互作用。结论·SPANXB的高表达与肝癌的预后呈负相关，其可能通过与MAD2L1、WDR5相互作用调控细胞周期并增强肝癌细胞的增殖活性。

关键词: 癌-睾丸抗原, SPANXB, 肝癌, 细胞周期

Abstract:

Objective ·To analyze the expression of cancer-testis antigen (CTA) family member SPANXB (sperm protein associated with the nucleus on the X chromosome B) in liver cancer and its correlation with the prognosis of liver cancer patients, and to explore the impact of SPANXB on liver cancer cell proliferation and its potential mechanism. Methods ·By using liver cancer sample data from the cancer genome atlas (TCGA) database, the expression of SPANXB in liver cancer tissue and its correlation with patient survival were analyzed. By constructing stable knockdown of SPANXB and stable overexpression of SPANXB in liver cancer cell lines, the effects of SPANXB on liver cancer cell proliferation were evaluated with live cell imaging experiments, EdU cell proliferation experiments and plate clone formation experiments. The regulatory pathways of SPANXB in liver cancer cell proliferation were explored through RNA-sequence (RNA-seq), and the effect of SPANXB on liver cancer cell cycle was validated through cell cycle experiments. Immunoprecipitation-mass spectrometry (IP-MS) was used to explore the proteins that interacted with SPANXB, and co-immunoprecipitation (Co-IP) was used to verify their interaction. Results ·The expression of SPANXB mRNA in liver cancer tissues was higher than that in normal tissues (P=0.003), and was negatively correlated with the survival of liver cancer patients. Stable knockdown of SPANXB could reduce the proliferation and clone formation ability of liver cancer cells, while stable overexpression of SPANXB could promote these processes. The analysis results of RNA-seq showed that knockdown of SPANXB could lead to downregulation of DNA replication and G1/S cell cycle transition-related pathways. The results of cell cycle experiments showed that knockdown of SPANXB could result in changes in the liver cancer cell cycle. The results of IP-MS and Co-IP showed that SPAXNB interacted with cell cycle-related proteins such as mitotic arrest defect 2-like protein 1 (MAD2L1) and WD repeat domain 5 (WDR5). Conclusion ·The high expression of SPANXB is negatively correlated with the prognosis of liver cancer. SPANXB may regulate the cell cycle and enhance the proliferation activity of liver cancer cells by interacting with MAD2L1 and WDR5.

Key words: cancer-testis antigen (CTA), sperm protein associated with the nucleus on the X chromosome B (SPANXB), liver cancer, cell cycle

中图分类号:

R735.7

薛煜, 张海龙, 雷鸣. 癌-睾丸抗原SPANXB在肝癌中的表达及其影响肝癌进展的机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(7): 801-813.

XUE Yu, ZHANG Hailong, LEI Ming. Expression of cancer-testis antigen SPANXB and its mechanism in affecting hepatocellular carcinoma progress[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(7): 801-813.

图/表 5

图1 SPANXB mRNA在泛癌中的表达以及LIHC患者 SPANXB mRNA表达与OS的相关性分析Note： A. Expression of SPANXB mRNA in pan-cancer. B. Expression of SPANXB mRNA in normal tissues and LIHC tissues. C. Expression of SPANXB mRNA in normal and tumor tissues obtained from the same patient. D. Proportion of SPANXB mRNA expression in normal tissues and LIHC tissues. E. OS analysis of LIHC patients based on high versus low SPANXB mRNA expression. F?H. OS analysis of LIHC Stage Ⅰ (F), Stage Ⅱ (G) and Stage Ⅲ+Ⅳ (H) patients based on high versus low SPANXB mRNA expression. ①P=0.000, ②P=0.004, ③P=0.013, ④P=0.045, ⑤P=0.003.

Fig 1 Analysis of SPANXB mRNA expression in pan-cancer and its correlation with OS in LIHC patients

图2 SPANXB在肝癌细胞系中的表达和HepG2细胞中敲低 SPANXB 对该细胞增殖表型的影响Note： A. Detection of SPANXB expression levels in 6 kinds of cells by Western blotting. B. Detection of SPANXB expression levels in HepG2 cells infected with sh-NC or sh-SPANXB lentivirus. C. Detection of relative proliferation capabilities of HepG2 cells infected with sh-NC or sh-SPANXB lentivirus by IncuCyteTM cell proliferation assay.D. Detection of DNA proliferation capacity of HepG2 cells infected with sh-NC or sh-SPANXB lentivirus by EdU assay. The blue areas represent the nucleus and the red areas represent a positive signal for EdU. E. Detection of clonogenic capacity of HepG2 cells infected with sh-NC or sh-SPANXB lentivirus by colony formation assay. ①P=0.000, compared with sh-NC.

Fig 2 Expression of SPANXB in hepatocellular carcinoma cell lines and effect of SPANXB knockdown on HepG2 cell proliferation phenotype

图3 Huh7细胞中过表达 SPANXB 及其对细胞增殖表型的影响Note： A. Detection of SPANXB expression levels in Huh7 cells overexpressing SPANXB by Western blotting. B. Detection of relative proliferation capabilities of Huh7 cells infected with Vector-Control or OE-SPANXB lentivirus by IncuCyteTM cell proliferation assay. C. Detection of DNA proliferation capacity of Huh7 cells infected with Vector-Control or OE-SPANXB lentivirus by EdU assay. The blue areas represent the nucleus and the red areas represent a positive signal for EdU. D. Detection of clonogenic capacity of Huh7 cells infected with Vector-Control or OE-SPANXB lentivirus by colony formation assay. ①P=0.014, compared with Vector-Control.

Fig 3 Overexpression of the SPANXB in Huh7 cells and its effects on cell proliferation phenotype

图4 SPANXB 表达与细胞增殖相关通路分析以及其对细胞周期的影响Note： A. Correlation analysis between SPANXB mRNA expression and enrichment scores of cell proliferation-related pathways in LIHC patient samples from TCGA database. B. Volcano plot of DEGs between HepG2 cells infected with sh-NC and sh-SPANXB lentivirus based on RNA-seq data. C. Heat map of DEGs between HepG2 cells infected with sh-NC and sh-SPANXB lentivirus. D. GO pathway analysis of upregulated and downregulated genes between HepG2 cells infected with sh-NC and sh-SPANXB lentivirus. E. GSEA enrichment analysis of G1/S cell cycle pathway genes in the RNA-seq data of HepG2 cells infected with sh-NC and sh-SPANXB lentivirus. F. Effect of SPANXB knockdown on cell cycle distribution. G. Effect of SPANXB overexpression on cell cycle distribution. ①P=0.039, ②P=0.003, ③P=0.170, ④P=0.017.

Fig 4 Analysis of SPANXB expression in relation to cell proliferation pathways and its impact on cell cycle

图5 SPANXB 过表达细胞的相互作用蛋白分析及验证Note： A. Detection of SPANXB in IP experiment by Western blotting. B. Volcano plot of the differential proteomic analysis between the experimental and control groups. C. Co-IP analysis of the interaction of SPANXB with MAD2L1. D. Co-IP analysis of the interaction of SPANXB with WDR5.

Fig 5 Analysis and validation of interaction proteins in SPANXB-overexpressed cells

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癌-睾丸抗原SPANXB在肝癌中的表达及其影响肝癌进展的机制研究

Expression of cancer-testis antigen SPANXB and its mechanism in affecting hepatocellular carcinoma progress

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