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*

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。

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

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.