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

doi:10.16431/j.cnki.1671-7236.2021.12.030

Abstract

Abstract: 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.

Key words: swine; Hepatitis E virus (HEV); ORF3 protein; HepG2 cells; riboflavin metabolism; lncRNA-mRNA regulatory network

CLC Number:

S852.65⁺1

JIAO Hanwei, ZHOU Zhixiong, LI Mengjuan, XIAO Yu, LI Bowen, GUO Xiaoyi, ZENG Hui, GU Guojing, SHUAI Xuehong. Identification of lncRNA-mRNA Regulatory Network of Riboflavin Metabolism in HepG2 Cells Affected by Swine Hepatitis E Virus ORF3 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4618-4627.

References

[1] SALINES M,ANDRAUD M,ROSE N.From the epidemiology of Hepatitis E virus (HEV) within the swine reservoir to public health risk mitigation strategies:A comprehensive review[J].Veterinary Research,2017,48(1):31.
[2] LI S S,HE Q,YAN L,et al.Infectivity and pathogenicity of different Hepatitis E virus genotypes/subtypes in rabbit model[J].Emerging Microbes & Infections,2020,9(1):2697-2705.
[3] DING Q,HELLER B,CAPUCCINO J M V,et al.Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles[J].Proceedings of the National Academy of Sciences of the United States of America,2017,114(5):1147-1152.
[4] NAN Y C,WU C Y,ZHAO Q,et al.Zoonotic Hepatitis E virus:An ignored risk for public health[J].Frontiers in Microbiology,2017,8:2396.
[5] HIMMELSBACH K,BENDER D,HILDT E.Life cycle and morphogenesis of the Hepatitis E virus[J].Emerging Microbes & Infections,2018,7(1):196.
[6] NAN Y C,WU C Y,ZHAO Q,et al.Vaccine development against zoonotic Hepatitis E virus:Open questions and remaining challenges[J].Frontiers in Microbiology,2018,9:266.
[7] MIRAZO S,GARDINALI N R,CECILIA D,et al.Serological and virological survey of Hepatitis E virus (HEV) in animal reservoirs from Uruguay reveals elevated prevalences and a very close phylogenetic relationship between swine and human strains[J].Veterinary Microbiology,2018,213:21-27.
[8] FERRE F,COLANTONI A,HELMER-CITTERICH M.Revealing protein-lncRNA interaction[J].Briefings in Bioinformatics,2016,17(1):106-116.
[9] HUANG Y.The novel regulatory role of lncRNA-miRNA-mRNA axis in cardiovascular diseases[J].Journal of Cellular and Molecular Medicine,2018,22(12):5768-5775.
[10] LIAO F,HUANG P C.Effects of moderate riboflavin deficiency on lipid metabolism in rats[J].Proceedings of the National Sciense Council,Republic of China.Part B,Life Sciences,1987,11(2):128-132.
[11] HIGASHI-OKAI K,NAGINO H,YAMADA K,et al.Antioxidant and prooxidant activities of B group vitamins in lipid peroxidation[J].Journal of UOEH,2006,28(4):359-368.
[12] WANG G G,LI W,LU X H,et al.Riboflavin alleviates cardiac failure in type Ⅰ diabetic cardiomyo-pathy[J].Heart International,2011,6(2):e21.
[13] MENG X J,PURCELL R H,HALBUR P G,et al.A novel virus in swine is closely related to the human Hepatitis E virus[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(18):9860-9865.
[14] REYES G R,PURDY M A,KIM J P,et al.Isolation of a cDNA from the virus responsible for enterically transmitted non-A,non-B hepatitis[J].Science,1990,247(4948):1335-1339.
[15] 陈书,卫飞雪,吴婷,等.器官移植病人戊型肝炎病毒慢性化感染及治疗[J].病毒学报,2015,31(3):293-298. CHEN S,WEI F X,WU T,et al.Chronic Hepatitis E virus infection and treatment in organ transplant recipients[J].Chinese Journal of Virology,2015,31(3):293-298.(in Chinese)
[16] KORKAYA H,JAMEEL S,GUPTA D,et al.The ORF3 protein of Hepatitis E virus binds to Src homology 3 domains and activates MAPK[J].Journal of Biological Chemistry,2001,276(45):42389-42400.
[17] ROY A K,KORKAYA H,OBEROI R,et al.The Hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase[J].Journal of Biological Chemistry,2004,279(27):28345-28357.
[18] ZAFRULLAH M,OZDENER M H,PANDA S K,et al.The ORF3 protein of Hepatitis E virus is a phosphoprotein that associates with the cytoskeleton[J].Journal of Virology,1997,71(12):9045-9053.
[19] PARVEZ M K,AL-DOSARI M S.Evidence of MAPK-JNK1/2 activation by Hepatitis E virus ORF3 protein in cultured hepatoma cells[J].Cytotechnology,2015,67(3):545-550.
[20] TYAGI S,KORKAYA H,ZAFRULLAH M,et al.The phosphorylated form of the ORF3 protein of Hepatitis E virus interacts with its non-glycosylated form of the major capsid protein,ORF2[J].Journal of Biological Chemistry,2002,277(25):22759-22767.
[21] TYAGI S,SURJIT M,ROY A K,et al.The ORF3 protein of Hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte[J].Journal of Biological Chemistry,2004,279(28):29308-29319.
[22] SURJIT M,OBEROI R,KUMAR R,et al.Enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101[J].Journal of Biological Chemistry,2006,281(12):8135-8142.
[23] 杜丽,成鹰,史巧芸,等.基因Ⅳ型戊型肝炎病毒ORF3对靶细胞脂肪因子表达的影响[J].动物医学进展,2013,34(6):78-80. DU L,CHENG Y,SHI Q Y,et al.Effect of ORF3 gene of Hepatitis E virus type Ⅳ on the expression of adipokines in target cells[J].Progress in Veterinary Medicine,2013,34(6):78-80.(in Chinese)
[24] DUTTA P,SEIRAFI J,HALPIN D,et al.Acute ethanol exposure alters hepatic glutathione metabolism in riboflavin deficiency[J].Alcohol,1995,12(1):43-47.
[25] CALDINELLI L,IAMETTI S,BARBIROLI A,et al.Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD[J].Protein Science,2008,17(3):409-419.
[26] MANTHEY K C,CHEW Y C,ZEMPLENI J.Riboflavin deficiency impairs oxidative folding and secretion of apolipoprotein B-100 in HepG2 cells,triggering stress response systems[J].Journal of Nutrition,2005,135(5):978-982.
[27] WAPINSKI O,CHANG H Y.Long noncoding RNAs and human disease[J].Trends in Cell Biology,2011,21(6):354-361.
[28] ZHANG K,ZHANG M X,YAO Q B,et al.The hepatocyte-specifically expressed lnc-HSER alleviates hepatic fibrosis by inhibiting hepatocyte apoptosis and epithelial-mesenchymal transition[J].Theranostics,2019,9(25):7566-7582.

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

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