转录组测序分析高尔基体蛋白73参与调控肝癌的作用机制

叶佩灵; 嘉红云; 彭亮

doi:10.3969/j.issn.1001-5256.2021.08.022

转录组测序分析高尔基体蛋白73参与调控肝癌的作用机制

DOI: 10.3969/j.issn.1001-5256.2021.08.022

1.
广州医科大学附属第二医院检验科, 广州 510260
2.
广州医科大学附属第五医院检验科, 广州 510700

基金项目:

广东省医学科学技术研究基金 (A2018153);

广州市卫生和计划生育科技项目 (20181A011072)

利益冲突声明: 本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明: 叶佩灵负责资料分析, 撰写论文; 嘉红云、彭亮负责课题设计, 指导文章撰写。

详细信息

通信作者:
嘉红云, hongyun_jia@163.com

中图分类号: R735.7
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- 被引次数: 0
出版历程
- 收稿日期: 2021-01-20
- 录用日期: 2021-04-12
- 出版日期: 2021-08-20

Mechanism of action of GP73 in the regulation of liver cancer: An analysis based on transcriptome sequencing

1.
Department of Clinical Laboratory, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
2.
Department of Clinical Laboratory, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, China

Research funding:

Medical Scientific Research Fund of Guangdong Province (A2018153);

Science and Technology Project of Guangzhou Health and Family Planning Commission (20181A011072)

摘要

摘要: 目的基于转录组分析技术, 探索高尔基体蛋白73(GP73)参与调控肝癌的作用机制。方法将肝癌细胞株Hep3B分为4组: GP73干扰组和对照组、GP73过表达组和对照组, 采用转录组测序技术检测4组样本mRNA的表达。根据测序数据筛选差异表达基因, 通过GO功能分析和KEGG富集分析差异表达基因。Western Blot实验验证信号通路相关蛋白的表达。符合正态分布的计量资料2组间比较采用t检验。结果通过分析Hep3B细胞干扰和过表达GP73后的差异表达基因, GO分析结果揭示了586个生物过程, GP73主要参与了细胞过程、单个组织的过程、生物调节等功能。KEGG分析表明GP73主要参与了PI3K-AKT、细胞因子/细胞因子受体相互作用、TNF、JAK-STAT等与肝癌密切相关的信号通路。Western Blot验证PI3K-AKT信号通路中的3个蛋白PI3K、p-AKT和AKT, 干扰组和过表达组的蛋白PI3K、p-AKT和对照组比较均有显著差异(P值均＜0.05)。结论通过转录组测序和生物信息分析筛选到GP73主要参与调控肝癌的信号通路, 为阐明GP73对肝癌的作用机制提供了新的思路。
- 肝肿瘤 /
- 转录组 /
- 信号传导
Abstract: Objective To investigate the mechanism of action of GP73 in the regulation of liver cancer based on transcriptome analysis. Methods Hepatoma Hep3B cells were divided into GP73 interference group, GP73 interference control group, GP73 overexpression group, and GP73 overexpression control group, and transcriptome sequencing was used to measure mRNA expression in the four groups. Differentially expressed genes were screened out based on sequencing data and were then analyzed by GO functional analysis and KEGG enrichment analysis. Western blot was used to verify the expression of signaling pathway-related proteins. The t-test was used for comparison of normally distributed continuous data between groups. Results The differentially expressed genes in Hep3B cells were analyzed after GP73 interference and overexpression. GO analysis revealed 586 biological processes, and GP73 was mainly involved in the processes such as cellular process, single-organism process, and biological regulation. KEGG analysis showed that GP73 was mainly involved in the signaling pathways closely associated with liver cancer, including the PI3K-AKT signaling pathway, the cytokine-cytokine receptor interaction signaling pathway, the TNF signaling pathway, and the JAK-STAT signaling pathway. Western blot was used to verify the three proteins PI3K, p-Akt, and Akt in the PI3K-AKT signaling pathway, and the results showed that there were significant differences in the expression of PI3K and p-Akt between the GP73 interference group and the GP73 interference control group and between the GP73 overexpression group and the GP73 overexpression control group (all P < 0.05). Conclusion The signaling pathways involving GP73 in the regulation of liver cancer are screened out by transcriptome sequencing and bioinformatics analysis, which provides new ideas for further elucidating the mechanism of action of GP73 in liver cancer.
- Liver Neoplasms /
- Transcriptome /
- Signal Transduction

HTML全文

图 1 干扰和过表达GP73后4个组的差异表达基因交叉分布情况

下载: 全尺寸图片幻灯片

图 2 干扰和过表达GP73后干扰组与过表达组差异表达基因的数目

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图 3 干扰和过表达GP73后差异表达基因分布图

注: 横坐标表示对照组样品基因表达量(取对数), 纵坐标表示实验组样品基因表达量(取对数)。实验组样品相对于对照组样品, 红点表示基因表达量上调的基因, 绿点表示基因表达量下调的基因, 蓝点表示基因表达量没有显著变化的基因。

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图 4 干扰和过表达GP73后共同差异表达基因的热图

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图 5 差异表达mRNA排名前10位的GO分析富集条目

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图 6 共同差异表达基因的KEGG分析排名前17位的条目

注: 纵坐标表示KEGG分析的条目, 横坐标表示显著差异表达基因的比例, 点的大小表示显著差异表达基因的数目, 点的颜色代表显著性, 表示不同的P值(0~1)。

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图 7 Western Blot验证PI3K、p-AKT、AKT基因在4组样本中的表达情况

注: *, P＜0.05。

下载: 全尺寸图片幻灯片

表 1 qRT-PCR引物序列表

基因名称	Primer名称	序列(5′-3′)	产物长度(bp)
GP73	GP73-F	GCCGGAGCCTCGAAAAGAG	190
	GP73-R	TTCCAGCTCCATGATCCGTG
GAPDH	GAPDH-F	GAGTCAACGGATTTGGTCGT	185
	GAPDH-R	GACAAGCTTCCCGTTCTCAG

下载: 导出CSV

参考文献(12)

[1]	PETRICK JL, FLORIO AA, ZNAOR A, et al. International trends in hepatocellular carcinoma incidence, 1978-2012[J]. Int J Cancer, 2020, 147(2): 317-330. DOI: 10.1002/ijc.32723.
[2]	LI JN, ZHENG RQ, LI N, et al. The biological characteristics of GP73 and its value in the diagnosis of liver fibrosis and cirrhosis[J]. J Clin Hepatol, 2019, 35(6): 1361-1364. DOI: 10.3969/j.issn.1001-5256.2019.06.040. 李佳娜, 郑瑞琦, 李娜, 等. 高尔基体蛋白73的生物学特征及在肝纤维化和肝硬化中的诊断价值[J]. 临床肝胆病杂志, 2019, 35(6): 1361-1364. DOI: 10.3969/j.issn.1001-5256.2019.06.040.
[3]	XIAO J, LONG F, PENG T, et al. Development and potential application of a simultaneous multiplex assay of Golgi protein 73 and alpha-fetoprotein for hepatocellular carcinoma diagnosis[J]. Eur Rev Med Pharmacol Sci, 2019, 23(8): 3302-3310. DOI: 10.26355/eurrev_201904_17692.
[4]	JIAO C, CUI L, PIAO J, et al. Clinical significance and expression of serum Golgi protein 73 in primary hepatocellular carcinoma[J]. J Cancer Res Ther, 2018, 14(6): 1239-1244. DOI: 10.4103/0973-1482.199784.
[5]	KE MY, WU XN, ZHANG Y, et al. Serum GP73 predicts posthepatectomy outcomes in patients with hepatocellular carcinoma[J]. J Transl Med, 2019, 17(1): 140. DOI: 10.1186/s12967-019-1889-0.
[6]	CHEN M, ZHAO K, LIU PC, et al. Effects of silencing GP73 with siRNA interference on the migration and invasion of human hepatoma HepG2 cells[J]. J Shanghai Jiaotong Univ (Med Sci), 2016, 36(11): 1588-1593. DOI: 10.3969/j.issn.1674-8115.2016.11.009. 陈默, 赵坤, 柳鹏程, 等. RNA干扰技术沉默GP73对人肝癌HepG2细胞迁移和侵袭的影响[J]. 上海交通大学学报(医学版), 2016, 36(11): 1588-1593. DOI: 10.3969/j.issn.1674-8115.2016.11.009.
[7]	KLADNEY RD, BULLA GA, GUO L, et al. GP73, a novel Golgi-localized protein upregulated by viral infection[J]. Gene, 2000, 249(1-2): 53-65. DOI: 10.1016/s0378-1119(00)00136-0.
[8]	YE PL, HE XX, WU XM. Expression of GP73 in liver tissue of primary liver cancer[J]. Guangdong Med J, 2014, 35(2): 234-236. DOI: 10.13820/j.cnki.gdyx.2014.02.025. 叶佩灵, 何欣欣, 吴晓蔓. 原发性肝癌组织中GP73蛋白的表达[J]. 广东医学, 2014, 35(2): 234-236. DOI: 10.13820/j.cnki.gdyx.2014.02.025.
[9]	YE PL, WU XM. Significance of GP73 level in primary hepatocellular carcinoma[J]. J Hainan Med Univ, 2014, 20(1): 83-86, 89. DOI: 10.13210/j.cnki.jhmu.2014.01.033. 叶佩灵, 吴晓蔓. 高尔基体糖蛋白73在原发性肝癌中的表达及意义[J]. 海南医学院学报, 2014, 20(1): 83-86, 89. DOI: 10.13210/j.cnki.jhmu.2014.01.033.
[10]	YE PL, WU XM. The significance of combined detection of serum Golgi protein 73, AFP, CEA, CA199 and GGT in the early diagnosis of primary hepatic carcinoma[J]. J Tropical Med, 2016, 16(4): 467-470. 叶佩灵, 吴晓蔓. 血清GP73联合AFP、CEA、CA199、GGT检测在PHC早期筛查的应用价值[J]. 热带医学杂志, 2016, 16(4): 467-470.
[11]	YANG Y, LIU Q, LI Z, et al. GP73 promotes epithelial-mesenchymal transition and invasion partly by activating TGF-β1/Smad2 signaling in hepatocellular carcinoma[J]. Carcinogenesis, 2018, 39(7): 900-910. DOI: 10.1093/carcin/bgy010.
[12]	YANG Y, LIU Q, ZHANG H, et al. Silencing of GP73 inhibits invasion and metastasis via suppression of epithelial-mesenchymal transition in hepatocellular carcinoma[J]. Oncol Rep, 2017, 37(2): 1182-1188. DOI: 10.3892/or.2017.5351.