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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (9): 10-19    DOI: 10.13523/j.cb.2103052
研究报告     
基于转录组测序探究ATP7B基因缺陷小鼠铜累积诱导肝细胞自噬的相关机制*
李潇瑾,李艳萌,李振坤,徐安健,杨晓曦,黄坚()
首都医科大学附属北京友谊医院科研实验中心 北京市临床医学研究所 北京 100050
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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摘要：

目的：分析ATP7B基因缺陷(Wilson's disease,WD)小鼠肝脏组织中自噬相关基因的表达和自噬相关蛋白的相互作用方式,探讨铜累积诱导肝内自噬活化的可能机制。方法：对4周龄和12周龄WD小鼠肝组织进行铜含量检测和转录组测序,对差异基因进行GO和KEGG富集分析,筛选自噬相关差异基因做qRT-PCR和Western blot验证,采用GeneMANIA数据库构建自噬相关差异蛋白的互作网络(PPI)并进行功能注释分析,抑制自噬相关蛋白的表达分析其对自噬的影响。结果：与野生型小鼠相比,WD小鼠肝铜含量显著升高,铜累积导致基因表达模式改变;基于GO数据库统计自噬相关差异基因数目,4周龄和12周龄分别有8个、51个,基于KEGG数据库统计,4周龄和12周龄分别有5个、19个;筛选Ulk1、Ddit4、Plk3等9个基因进行qRT-PCR,定量结果与测序结果表达趋势基本一致;其编码的蛋白质通过共表达、共定位等方式互相作用;Western blot结果显示铜累积导致Ulk1、Plk3、Park2蛋白表达显著增加和细胞自噬发生,抑制Ulk1、Plk3、Park2的蛋白质表达可显著下调细胞自噬水平。结论：WD不同阶段的铜累积可调节肝脏多个自噬相关基因的表达,通过其编码的自噬相关蛋白的互相作用共同诱导肝脏自噬活化以缓解肝损伤。
关键词： ATP7B;  威尔逊病;  肝豆状核变性;  铜累积;  自噬    
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
收稿日期: 2021-03-21 出版日期: 2021-09-30
ZTFLH:  Q812  
基金资助: * 国家自然科学基金(81602032);北京市优秀人才项目(2016000021469G224)
通讯作者: 黄坚     E-mail: huangj1966@hotmail.com
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李潇瑾
李艳萌
李振坤
徐安健
杨晓曦
黄坚

引用本文:

李潇瑾,李艳萌,李振坤,徐安健,杨晓曦,黄坚. 基于转录组测序探究ATP7B基因缺陷小鼠铜累积诱导肝细胞自噬的相关机制*[J]. 中国生物工程杂志, 2021, 41(9): 10-19.

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.

链接本文:

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

基因 引物序列
Ulk1 F: GCATCGAGCAAAACCTGCAA
R: GGGGAGAAGGTGTGTAGGGA
Ddit4 F: CTGGCATACAGTGAGCCGTG
R: AGGGTCAACTGAAAGGTGGG
Mtmr4 F: AAGTTTGGAGACCGCTGTGG
R: GATGCAGGACCAGAAATGCTTC
Rnf152 F: CTGCCCATCTCTAAGGAGCG
R: ATGTGGAGCTCTTCACCACG
Park2 F: GGGATTCAGAAGCAGCCAGA
R: GAGGGTTGCTTGTTTGCAGG
Npc1 F: GCCACAGAAGGCGGTACTTTG
R: GAACATGCGCCTCAGACAGT
Plk3 F: TCCTGCTTGGCTCCTGTAGTT
R: GGCATGAAGGCCACACAGTT
Prkaa2 F: ACTCTGCTGATGCACATGCT
R: TCGTAGGAGGGGTCTTCAGG
Atg16l2 F: ACAGGTGTTCAGGGCAGATG
R: CATTAACAGCAGTGCAGTGGG
GAPDH F: TGGCCTTCCGTGTTCCTAC
R: GAGTTGCTGTTGAAGTCGCA
表1  用于qRT-PCR检测的基因引物
图1  ATP7B基因敲除对小鼠肝铜含量的影响
图2  差异表达基因的聚类热图
图3  自噬相关差异表达基因的维恩图
项目 基因 4周 12周
log2FC P log2FC P
KD vs WT Ulk1 1.14 2.13×10-8 2.64×10-2 0.91
Ddit4 -1.64 7.40×10-4 2.10 1.47×10-21
Mtmr4 -1.18 1.68×10-4 -1.03 4.91×10-8
Rnf152 1.13 3.68×10-6 -2.53 3.00×10-10
Park2 1.02 2.97×10-2 0.68 4.01×10-2
Npc1 1.30 2.71×10-6 -0.61 9.42×10-4
Plk3 2.37×10-2 0.95 1.10 1.00×10-3
Prkaa2 0.19 0.30 -1.53 5.36×10-14
Atg16l2 -0.25 0.41 1.17 1.83×10-3
表2  自噬相关差异表达基因测序结果
图4  自噬相关差异基因的qRT-PCR验证
图5  自噬相关差异蛋白的PPI网络与功能注释
图6  过量铜处理诱导小鼠肝癌细胞内自噬相关差异蛋白的表达和自噬活化
图7  抑制自噬相关蛋白Ulk1、Plk3、Park2的表达对细胞自噬的影响
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