猪戊型肝炎病毒ORF3蛋白影响HepG2细胞核黄素代谢的lncRNA-mRNA调控网络的鉴定

Identification of lncRNA-mRNA Regulatory Network of Riboflavin Metabolism in HepG2 Cells Affected by Swine Hepatitis E Virus ORF3 Protein

为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。

In order to preliminarily identify and explore the lncRNA-mRNA regulatory network of ORF3 protein of swine Hepatitis E virus(HEV)affecting the riboflavin metabolism signal pathway of HepG2,in this study,we constructed the Adenovirus overexpression vector to prepare the high titer Adenovirus,and mediated the overexpression of swine HEV-ORF3 in HepG2 cells.After the overexpression of ORF3 protein was verified by Western blotting,lncRNA high-throughput sequencing was used to screen the differentially expressed lncRNA and predict the targeted genes.GO function and KEGG pathway enrichment analysis was performed for the differentially expressed lncRNA targeted genes,and the lncRNA-mRNA regulatory network related to riboflavin metabolism signal pathway was preliminarily identified.Western blotting showed that the target band appeared at about 12 ku,indicating the successful overexpression of swine HEV-ORF3 in HepG2 cells mediated by Adenovirus.High throughput sequencing of lncRNA showed that 102 differentially expressed lncRNAs were up-regulated and 80 differentially expressed lncRNAs were down regulated.GO function and KEGG pathway enrichment found that lncRNA(MSTRG.13995.2)and lncRNA(MSTRG.1960.1)might be differentially expressed lncRNAs related to riboflavin metabolism signaling pathway,which affected riboflavin metabolism signal pathway by cis regulating their target genes APC-5 and FLAD1,respectively.The results showed that lncRNA(MSTRG.13995.2)-APC5 and lncRNA(MSTRG.1960.1)-FLAD1 might be the lncRNA-mRNA regulatory network that affected the riboflavin metabolism signal pathway of HepG2.