Study On The Expression Profile Of CircRNAs In HepG2 Cells Affected By ORF3 Protein Of Porcine Hepatitis E Virus

Swine Hepatitis E(SHE)is a new zoonotic infectious disease caused by Swine Hepatitis E Virus(SHEV)infection.Open reading frame 3(ORF3)is a key regulatory and toxic protein of SHEV.Circular RNA(circular RNA,circRNA)is a special non-coding RNA molecule with a closed circular structure,widely present in eukaryotic cells.Studies have shown that circRNA molecules are rich in miRNA binding sites,which can be used as miRNA molecular sponges to competitively bind miRNA target sites,reduce the binding of target genes to corresponding miRNAs,and regulate downstream mRNA expression levels;circRNA can The interaction of disease-related miRNAs plays an important role in the disease process.In order to explore the mechanism of SHEV ORF3 affecting target cells,this topic carried out the following experiments:1.SHEV ORF3 gene overexpression recombinant adenovirus packaging and infectionThe SHEV ORF3 gene fragment of genotype IV was synthesized by Shanghai Shengong Biological Engineering Co.,Ltd.and the coding region was 345 bp.Design upstream and downstream primers according to the sequence published by Gen Bank,and design EcoRI and BamHI as the cleavage sites for the upstream and downstream primers respectively,and clone the type IV SHEV ORF3 gene into adenovirus by Polymerase Chain Reaction(PCR)technology In the shuttle plasmid ADV4,the recombinant adenovirus shuttle plasmid ADV4-SHEV-ORF3 was constructed and co-transfected with the backbone plasmid pGP-Ad-Pac Vector into HEK293A cells.The cells and culture medium were collected and the recombinant adenovirus ADV4-was obtained by repeated freezing and thawing and centrifugation ORF3 and control recombinant adenovirus ADV4-GFP;the recombinant adenovirus was amplified to prepare high-titer recombinant adenoviruses ADV4-ORF3 and ADV4-GFP,and the virus titer was detected by the method of trace whole cell disease.The HepG2 cells were plated at 1×10~6cells/well on a 12-well cell culture plate,and the recombinant adenoviruses ADV4-ORF3 and ADV4-GFP were added for 24h according to the multiplicity of infection(MOI)=5:1,and inverted fluorescence was used for 24h Microscope to detect the expression of GFP-ORF3 fusion protein;for ORF3 gene sequence,use NCBI primer to design qRT-PCR primers online,using GAPDH as internal reference,use comparative Ct value method,use qRT-PCR to detect the expression of GFP-ORF3 fusion protein mRNA level.Results:The titer of the recombinant adenovirus ADV4-ORF3 was about 5×10~8PFU/m L;cells infected with the recombinant adenoviruses ADV4-ORF3 and ADV4-GFP basically showed green fluorescence;qRT-PCR detection results showed that in ADV4-ORF3 In the infected group,the relative expression of ORF3 was significantly higher than that of the ADV4-GFP control group,and the difference was about 1500 times,indicating that adenovirus successfully mediated the overexpression of ORF3 protein in HepG2 cells.2.Analysis of circRNA expression profile of SHEV ORF3 mediated differential expression in HepG2 cells.After the SHEV ORF3 protein was successfully overexpressed in HepG2 cells,the total RNA of ADV4-ORF3 and ADV4-GFP infected HepG2 cells was extracted separately,and the expression profile of differentially expressed circRNAs was analyzed using high-throughput sequencing technology.The number of back-splicing junctions(BS)in the loop,the differential expression of top10 circRNAs,and verification using qRT-PCR;using miRanda and Targetscan software to analyze the circRNA-hosting gene;using GO and KEGG,the difference The expressed circRNA was analyzed for functional enrichment.Results:Compared with the ADV4-GFP infection group,a total of 1105 circRNAs with up-regulation and 1556 down-regulation were identified in the ADV4-ORF3infection group.The qRT-PCR results confirmed that up-regulated hsa＿circ＿0001423,hsa＿circ＿0006404 and down-regulated hsa＿circ＿0004833 hsa＿circ＿0007444 is consistent with the sequencing results,indicating that the high-throughput sequencing data is reliable;GO functional enrichment analysis shows that the significant differential expression of circRNAs is mainly involved in cellular components,biological processes,and molecular functions(molecular function);KEGG pathway enrichment analysis showed that the significantly differentially expressed circRNAs mainly participated in the top20 pathway.Among them,the SHEV ORF3 protein was initially screened out and the 4 significantly differently expressed circRNAs associated with the riboflavin metabolism(ko00740)pathway in HepG2 cells One mRNA is circRNA13511,ciRNA140,hsa＿circ＿0130711,hsa＿circ＿0130715and ENST00000358229.Conclusion:This study used adenovirus-mediated overexpression of SHEV ORF3in HepG2 cells for the first time,excavated the expression profiles of 1105 up-regulated and 1556 down-regulated circRNAs;and preliminary analysis showed that SHEV ORF3has a significant correlation with HepG2 cell riboflavin metabolism.Differentially expressed circRNA13511,ciRNA140,hsa＿circ＿0130711,hsa＿circ＿0130715,and ENST00000358229,which may regulate the process of lipid metabolism of target cells,provide clues for the function of SHEV ORF3 protein,and lay a foundation for further revealing the molecular mechanism of SHEV infection and preventing and treating SHE.