Transcriptome Analysis Of HepG2 Stable Cell Line With Expression Of SHEV ORF3

HEV-mediated hepatitis has become a global public health problem, having been impacted on human life seriously. SHEV, the swine form of HEV, has been identified in pigs. As a regulatory protein, ORF3 protein influences multiple signal pathways of target cells.To shed light on the effected mechanism of SHEV ORF3 on the target cells. In our first study, based on the sequence of SHEV ORF3, a pair of up and down stream primer was designed. We first cloned genotype IV HEVORF3 gene from pET42a-ORF3 by PCR. Constructed pEGFP-ORF3 and pEGFP lentiviral vector. Secondly, we packed the lentiviral vector in 293T cells in order to infect HepG2 cells. Thirdly, we obtained the stable cell lines in which pEGFP-ORF3 and pEGFP were stablely expressed, and which were identified by flow cytometry and Western blotting. We successfully constructed the HepG2-pEGFP-ORF3 stable expression cell lines.The next the total RNA from HepG2-pEGFP-ORF3 stably expressing cell lines was extracted by Trizol reagent, and sequenced by the high-throughput RNA-Seq-based screen, which was further validated with quantitative real-time PCR was performed to identify the differential expression genes in HepG2-pEGFP-ORF3 cells, and Gene Ontology were performed to assign the differentially expressed genes. The results indicated that in the established HepG2-pEGFP-ORF3 cells, the mRNA levels CLDN6?YLPM1?APOC3? NLRP1?SCARA3?FGA?FGG?FGB and FREM1 were up-regulated, whereas SLC2A3? DKK1?BPIFB2 and PTGR1 were down-regulated. Among them, the expression deregulation of CLDN6 and FREM1 might contribute to the deregulation of integral membrane protein and basement membrane protein expression; the expression deregulation of NLRP1 might affects the apoptosis of HepG2 cells, the expression deregulation of PTGR1 might affects the process apoptosis of HepG2 cells. Particularly, the expression deregulation of APOC3?SCARA3 and DKK1, might affects the lipid metabolism of the HepG2 cells. The findings provide scientific clues to further understand the function of SHEV ORF3 and elucidate the pathogenesis mechanism of HEV.