Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing

Mengni ZHANG; Xiaoxun ZHANG; Jiafeng OUYANG; Jin CHAI; Xiaoling WU

doi:10.16016/j.2097-0927.202303158

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Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing

Basic Medicine | 更新时间：2026-01-09

- Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing
- Journal of Army Medical University Vol. 45, Issue 18, Pages: 1883-1893(2023)
- 作者机构：
  
  1.610031 成都，西南交通大学医学院
  2.400038 重庆，陆军军医大学(第三军医大学)第一附属医院消化内科，胆汁淤积肝病中心和代谢性肝病中心
  3.610083 成都，西部战区总医院消化内科
- 作者简介：
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(82000545);the General Program of National Natural Science Foundation of China(81770583)
- DOI：10.16016/j.2097-0927.202303158
  CLC： R322.47;R394-33;R394.3
- Received：26 April 2023，
  
  Revised：2023-06-01，
  
  Published：30 September 2023
- 稿件说明：
移动端阅览
Mengni ZHANG, Xiaoxun ZHANG, Jiafeng OUYANG, et al. Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing[J]. Journal of Army Medical University, 2023, 45(18): 1883-1893.
DOI：

Mengni ZHANG, Xiaoxun ZHANG, Jiafeng OUYANG, et al. Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing[J]. Journal of Army Medical University, 2023, 45(18): 1883-1893. DOI： 10.16016/j.2097-0927.202303158.

摘要

目的

通过转录组测序分析结合型胆汁酸诱导肝细胞的基因表达情况，筛选和验证结合型胆汁酸诱导肝细胞代谢的Hub基因。

方法

使用牛磺胆酸(taurocholate acid，TCA)处理野生型小鼠的原代肝细胞，转录组测序(RNA sequencing

RNA-Seq)检测经TCA诱导后肝细胞的基因表达情况，将Fold Change≥1.5且

0.05作为筛选标准获取差异表达基因(differentially expressed genes

DEGs)并进行生物信息学分析。使用STRING(search tool for the retrieval of interesting genes)数据库建立DEGs的蛋白质-蛋白质互作网络(protein-protein interaction，PPI)，基于PPI网络利用Cytoscape软件找寻Hub基因。利用实时荧光定量PCR(RT-qPCR)在体内外水平对目标Hub基因进行验证，使用蛋白质免疫印迹试验检测肝细胞内PSAT1/AKT/GSK3β信号通路相关蛋白的表达，使用糖原检测试剂盒检测肝细胞和肝组织糖原水平。

结果

TCA处理小鼠原代肝细胞共有差异基因293个，其中上调基因122个，下调基因171个。基因本体(Gene Ontology

GO)功能富集分析、京都基因与基因百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路富集分析及基因集富集分析(Gene Set Enrichment Analysis，GSEA)的

结果

显示，DEGs主要富集在代谢、炎症、肿瘤等通路。使用Cytohubba插件得到16个Hub基因，包括

Ccnd

1、

Ppara

、

Irs

1、

Ccl

2、

Ddit

3、

Serpine

1、

2、

Atf

3、

Phgdh

、

Trib

3、

Lepr

、

Psat

1、

Cxcl

2、

Asns

、

Fgf

21。RT-qPCR检测

结果

显示，多种结合型胆汁酸包括TCA、甘氨鹅去氧胆酸(glycochenodeoxycholic acid

GCDCA)、甘氨胆酸(glycocholic acid hydrate

GCA)均能诱导小鼠原代肝细胞内

Psat

1 mRNA表达水平增高(

＜0.05)。此外，胆管结扎(bile duct ligation

BDL)胆汁淤积小鼠肝脏中的

Psat

1 mRNA水平增高(

＜0.05)。Western blot检测结果显示，TCA能够诱导肝细胞内

Psat

1表达及激活AKT/GSK3β信号通路。TCA能够诱导小鼠原代肝细胞糖原合成增加，与BDL小鼠模型肝组织糖原合成增加以及血糖水平降低的结果一致(

＜0.05)。

结论

Psat

1可能是胆汁酸影响肝细胞糖代谢的相关基因，其高水平表达可能与肝细胞糖代谢的改变有关。

Abstract

Objective

To screen and verify the Hub genes involved in conjugated bile acid-induced hepatocyte metabolism through transcriptome sequencing analysis.

Methods

Primary mouse hepatocytes were treated with taurocholic acid (TCA)

and the gene expression of TCA-induced hepatocytes was detected by RNA-Seq analysis. Differential expression genes (DEGs) were identified using a fold change ≥1.5 and

value

0.05 as the screening criteria. The protein-protein interaction (PPI) network of DEGs was constructed using the STRING database

and Hub genes were screened using Cytoscape software based on the PPI network. Real-time qPCR was used to validate the identified Hub gene

vitro

and

vivo

. Western blotting was employed to detect the expression of proteins related to PSAT1/AKT/GSK3β signaling pathway. The glycogen level in the hepatocytes and liver tissues were detected using glycogen dete

ction kit.

Results

RNA-Seq analysis identified 293 DEGs in primary mouse hepatocytes upon TCA treatment

including 122 up-regulated genes and 171 down-regulated genes. Gene Ontology (GO) analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis

and Gene Set Enrichment Analysis (GSEA) revealed that these DEGs were mainly enriched in metabolism

inflammatory and tumorigenic pathways. Cytohubba plugin identified 16 Hub genes

including

Ccnd

Ppara

Irs

Ccl

Ddit

Serpine

Atf

Phgdh

Trib

Lepr

Psat

Cxcl

Asns

and

Fgf

21. RT-qPCR results demonstrated that various conjugated bile acids

glycocholic acid (GCA)

glycochenodeoxycholic acid (GCDCA)

and TCA can induce a significant increase in

Psat

1 mRNA level in primary mouse hepatocytes (

0.05).

vivo

experiments also showed that the mRNA level of

Psat

1 was increased significantly in the liver tissues of mouse cholestatic model induced by bile duct ligation (BDL) when compared to the control group (

0.05). Western blotting indicated that TCA treatment induced the expression of

Psat

1 and activation of AKT/GSK3β signaling pathway in primary mouse hepatocytes. TCA treatment also resulted in an increase in glycogen synthesis in primary mouse hepatocytes

which was consistent with an increase in glycogen synthesis in the liver tissue and a decrease in blood glucose level in the mouse model established with BDL (

0.05).

Conclusion

Psat

1 may be a gene involved in alteration of

glucose metabolism induced by bile acid

and its high expression may be associated with these changes in glucose metabolism in hepatocytes.

关键词

Keywords

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