The Mechanism of Copper Accumulation Induced Autophagy in Hepatocytes of <i>ATP7B</i>-deficient Mice Based on RNA-sequencing

doi:10.13523/j.cb.2103052

China Biotechnology

2021, Vol. 41

Issue (9): 10-19 DOI: 10.13523/j.cb.2103052

The Mechanism of Copper Accumulation Induced Autophagy in Hepatocytes of ATP7B-deficient Mice Based on RNA-sequencing

LI Xiao-jin,LI Yan-meng,LI Zhen-kun,XU An-jian,YANG Xiao-xi,HUANG Jian(

)

Experimental Center, Beijing Friendship Hospital, Capital Medical University, Beijing Clinical Medicine Institute, Beijing 100050, China

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Abstract

Objective:To investigate the expression of autophagy-related genes and the interaction of autophagy-related proteins in liver tissues of ATP7B-deficient (WD) mice, and to explore the possible mechanism of copper accumulation induced autophagy activation in liver.Methods:The liver copper content of 4 weeks and 12 weeks of WD mice was detected. RNA-sequencing of liver tissues was conducted, and the GO and KEGG pathways of differentially expressed genes were analyzed by bioinformatics. The expression of autophagy-related differentially expressed genes was detected by qRT-PCR and Western blot. GeneMANIA database was used to construct the protein-protein interaction network (PPI) which was related to these autophagy-related proteins, and functional annotation was carried out to analyze its autophagy-related biological function and protein interactions. The expression of autophagy-related proteins was inhibited to analyze its effect on autophagy.Results:Compared with wild-type mice, liver copper content of WD mice was significantly increased, and the copper accumulation led to changes in gene expression pattern. According to the GO database, the number of autophagy-related differential genes in WD mice was 8 at 4 weeks and 51 at 12 weeks. According to KEGG database, the number of autophagy-related differential genes was 5 at 4 weeks and 19 at 12 weeks, respectively. Nine genes, including Ulk1, Ddit4 and Plk3, were screened for qRT-PCR, and the quantitative results was basically consistent with the sequencing results. These autophagy-related proteins interact with each other through co-expression and co-localization. Western blot results showed that copper accumulation significantly increased the protein expressions of Ulk1, Plk3 and Park2, and resulted in autophagy. Inhibition of Ulk1, Plk3 and Park2 expression significantly down-regulated the level of autophagy.Conclusion:Copper accumulation at different stages of WD can regulate the expression of several autophagy-related genes in the liver, and the liver autophagy activation was induced by the interaction of autophagy-related proteins which could alleviate liver injury of WD.

Key words： ATP7B Wilson's disease Hepatolenticular degeneration Copper accumulation Autophagy

Received: 21 March 2021 Published: 30 September 2021

ZTFLH:

Q812

Corresponding Authors: Jian HUANG E-mail: huangj1966@hotmail.com

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	Xiao-jin LI
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Cite this article:

LI Xiao-jin,LI Yan-meng,LI Zhen-kun,XU An-jian,YANG Xiao-xi,HUANG Jian. The Mechanism of Copper Accumulation Induced Autophagy in Hepatocytes of ATP7B-deficient Mice Based on RNA-sequencing. China Biotechnology, 2021, 41(9): 10-19.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103052 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I9/10

Table 1 Gene primers for qRT-PCR detection

Fig.1 Effect of ATP7B gene knockout on liver copper content in mice The 4-week-old and 12-week-old ATP7B gene knockout (KD) mice and wild-type (WT) mice were sacrificed and the liver tissues were ground, respectively. The copper concentration in the liver tissue grinding solution was detected by copper quantitative detection kit (n=5; ** P<0.01)

Fig.2 Clustering heat map of differentially expressed genes Cluster maps of liver tissue sequencing of 4-week-old and 12-week-old ATP7B knockout (KD) mice and wild-type (WT) mice. Three mice were in each group. The numbers on the right of the illustration are multiples of up-regulated or down-regulated gene expression

Fig.3 Venn diagram of autophagy-related differentially expressed genes The autophagy-related differentially expressed genes of KD vs WT groups at 4 and 12 weeks of age were analyzed based on GO(a) and KEGG(b) databases

Table 2 RNA-seq results of autophagy-related differentially expressed genes

Fig.4 RNA expression of autophagy-related differential genes by qRT-PCR The RNA expression of autophagy-related genes in liver tissues of 4-week-old and 12-week-old ATP7B gene knockout (KD) mice and wild-type (WT) mice detected by qRT-PCR (n=3; * : P<0.05; ** : P<0.01)

Fig.5 PPI network and functional annotation of autophagy-related differential proteins

Fig.6 Excessive copper treatment induced the expression of autophagy-related differential proteins and autophagy activation in mouse hepatoma cells The expression of Ulk1, Plk3, Park2, Beclin-1, P62 proteins in copper treated H22 cells were detected by Western blot

Fig.7 The effects of Ulk1-siRNA, Plk3-siRNA, Park2-siRNA on cell autophagy (a) The expression of Beclin-1 and P62 proteins in Ulk1-siRNA treated H22 cells were detected by Western blot (b) The expression of Beclin-1 and P62 proteins in Plk3-siRNA treated H22 cells were detected by Western blot (c) The expression of Beclin-1 and P62 proteins in Park2-siRNA treated H22 cells were detected by Western blot


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