猪圆环病毒2型感染PK-15细胞差异表达miRNA和mRNA互作网络
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摘要
旨在筛选猪圆环病毒2型感染猪肾上皮细胞(PK-15)后差异表达miRNA和mRNA,并探究miRNA和mRNA之间的互作关系。使用茎环定量PCR检测多个热点miRNA在PCV2感染组和未接种病毒组之间的差异表达水平以发现差异表达miRNA,分析PCV2感染后第12小时的蛋白质组学研究结果(GEO登录号:GSE71945)以获得差异表达mRNA,并分析差异表达miRNA和mRNA之间的互相作用关系。结果显示,在PCV2感染后第12小时miR-10b、miR-128、miR-155-5p、miR-21、miR-29b、miR-361-3p等表达量显著性变化,使用miRecords预测其靶基因,分别发现7 105、9 741、7 451、7 183、7 971、8 793个靶基因;PCV2感染后第12小时共有158个差异表达mRNA,分属于多种细胞结构成分并参与多条重要的细胞生物途径,且差异miRNA的靶基因和差异mRNA之间有大量的交集基因。上述结果表明,PCV2感染能导致细胞内多种miRNA差异表达,造成miRNA的靶基因表达紊乱,进而影响细胞正常的生命进程。
The purpose of this study was to elucidate changes in miRNA and mRNA expression profiles of PCV2 infected PK-15 cells,and to illuminate the interaction between differentially expressed miRNAs and mRNAs.Stem-loop quantitative PCR was used to detect and identify differential expression levels of multiple hot spot miRNAs in PCV2 infected and uninfected groups.The results of proteomic study of PCV2 infected cells at the 12 th hour post infection(hpi)were analyzed(GEO Accession No.:GSE71945)to obtain differentially expressed mRNA and to analyze the interaction between differentially expressed miRNA and mRNA.It was found that miR-10 b,miR-128,miR-155-5p,miR-21,miR-29 b,miR-361-3p were differentially expressed at 12 hpi.By using miRecords to predict their target genes,7 105,9 741,7 451,7 183,7 971and8 793 target genes were found,respectively.Totally there were 158 differentially expressed mRNAs that belonged to various cell components and participated in many important biological pathways,and there was a large number of common target genes between the differentiallyexpressed miRNAs candidate target genes and differentially expressed mRNAs.In summary,PCV2 infection can lead to disruption of multiple miRNA expression in cells,resulting in disorganization of miRNA target genes,which in turn influences the normal life events of cells.
The purpose of this study was to elucidate changes in miRNA and mRNA expression profiles of PCV2 infected PK-15 cells,and to illuminate the interaction between differentially expressed miRNAs and mRNAs.Stem-loop quantitative PCR was used to detect and identify differential expression levels of multiple hot spot miRNAs in PCV2 infected and uninfected groups.The results of proteomic study of PCV2 infected cells at the 12 th hour post infection(hpi)were analyzed(GEO Accession No.:GSE71945)to obtain differentially expressed mRNA and to analyze the interaction between differentially expressed miRNA and mRNA.It was found that miR-10 b,miR-128,miR-155-5p,miR-21,miR-29 b,miR-361-3p were differentially expressed at 12 hpi.By using miRecords to predict their target genes,7 105,9 741,7 451,7 183,7 971and8 793 target genes were found,respectively.Totally there were 158 differentially expressed mRNAs that belonged to various cell components and participated in many important biological pathways,and there was a large number of common target genes between the differentiallyexpressed miRNAs candidate target genes and differentially expressed mRNAs.In summary,PCV2 infection can lead to disruption of multiple miRNA expression in cells,resulting in disorganization of miRNA target genes,which in turn influences the normal life events of cells.
引文
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[2]HARDING J C S,CLARK E G.Recognizing and diagnosing postweaning multisystemic wasting syndrome(PMWS)[J].Swine Health Prod,1997,5(5):201-203.
[3]RAMOS-VARA J A,DURAN O,RENDER J A,et al.Porcine dermatitis and nephropathy syndrome in the USA[J].Vet Rec,1997,141(18):479-480.
[4]SZEREDI L,D N A,SOLYMOSI N,et al.Association of porcine circovirus type 2with vascular lesions in porcine pneumonia[J].Vet Pathol,2012,49(2):264-270.
[5]BARTEL D P.MicroRNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[6]BARTEL D P.MicroRNAs:Target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[7]GRUNDHOFF A,SULLIVAN C S.Virus-encoded microRNAs[J].Virology,2011,411(2):325-343.
[8]NU′EZ-HERN NDEZ F,P REZ L J,MUOZM,et al.Identification of microRNAs in PCV2subclinically infected pigs by high throughput sequencing[J].Vet Res,2015,46:18.
[9]NU′EZ-HERN NDEZ F,P REZ L J,VERA G,et al.Evaluation of the capability of the PCV2genome to encode miRNAs:Lack of viral miRNA expression in an experimental infection[J].Vet Res,2015,46:48.
[10]HONG J S,KIM N H,CHOI C Y,et al.Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins[J].Vet Res,2015,46:39[2018-10-01].https://doi.org/10.1186/s13567-015-0172-5.
[11]ZHANG P,WANG L Y,LI Y P,et al.Identification and characterization of microRNA in the lung tissue of pigs with different susceptibilities to PCV2infection[J].Vet Res,2018,49(1):18.
[12]QUAN R,WEI L,ZHU S S,et al.Involvement of miR-15ain G0/G1phase cell cycle arrest induced by porcine circovirus type 2replication[J].Sci Rep,2016,6:27917.
[13]CHENG S,ZHANG M,LI W T,et al.Proteomic analysis of porcine alveolar macrophages infected with porcine circovirus type 2[J].J Proteomics,2012,75(11):3258-3269.
[14]ZHANG X,ZHOU J Y,WU Y P,et al.Differential proteome analysis of host cells infected with porcine circovirus type 2[J].J Proteome Res,2009,8(11):5111-5119.
[15]LIU J,BAI J,LU Q,et al.Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification(iTRAQ)labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2[J].J Proteomics,2013,79:72-86.
[16]LIU J,BAI J,ZHANG L L,et al.Proteomic alteration of PK-15cells after infection by porcine circovirus type 2[J].Virus Genes,2014,49(3):400-416.
[17]FANG W J,LIN C Z,ZHANG H H,et al.Detection of let-7amicroRNA by real-time PCR in colorectal cancer:A single-centre experience from China[J].J Int Med Res,2007,35(5):716-723.
[18]OPRIESSNIG T,MENG X J,HALBUR P G.Porcine circovirus type 2-associated disease:Update on current terminology,clinical manifestations,pathogenesis,diagnosis,and intervention strategies[J].JVet Diagn Invest,2007,19(6):591-615.
[19]SEGAL S J.Porcine circovirus type 2(PCV2)infections:Clinical signs,pathology and laboratory diagnosis[J].Virus Res,2012,164(1-2):10-19.
[20]RAMAMOORTHY S,MENG X J.Porcine circoviruses:A minuscule yet mammoth paradox[J].Anim Health Res Rev,2009,10(1):1-20.
[21]KRACHT M,SAKLATVALA J.Transcriptional and post-transcriptional control of gene expression in inflammation[J].Cytokine,2002,20(3):91-106.
[22]ZHOU B Y,WANG S,MAYR C,et al.miR-150,a microRNA expressed in mature B and T cells,blocks early B cell development when expressed prematurely[J].Proc Natl Acad Sci U S A,2007,104(17):7080-7085.
[23]CEPPI M,PEREIRA P M,DUNAND-SAUTHIERI,et al.MicroRNA-155 modulates the interleukin-1signaling pathway in activated human monocyte-derived dendritic cells[J].Proc Natl Acad Sci U S A,2009,106(8):2735-2740.
[24]LU L F,LISTON A.MicroRNA in the immune system,microRNA as an immune system[J].Immunology,2009,127(3):291-298.
[25]WANG X M,XU X L,WANG W,et al.MicroR-NA-30a-5p promotes replication of porcine circovirus type 2through enhancing autophagy by targeting 14-3-3[J].Arch Virol,2017,162(9):2643-2654.
[26]CHEN W,WANG J,LIU S L,et al.MicroRNA-361-3p suppresses tumor cell proliferation and metastasis by directly targeting SH2B1in NSCLC[J].JExp Clin Cancer Res,2016,35:76.
[27]ROTH C,STCKRATH I,PANTEL K,et al.Low levels of cell-free circulating miR-361-3p and miR-625*as blood-based markers for discriminating malignant from benign lung tumors[J].PLoS One,2012,7(6):e38248.
[28]LIU H,CHENG X H.MiR-29breverses oxaliplatinresistance in colorectal cancer by targeting SIRT1[J].Oncotarget,2018,9:12304-12315.
[29]HUAN Y J,ZHU J,WANG H M,et al.Epigenetic modification agents improve genomic methylation reprogramming in porcine cloned embryos[J].J Reprod Dev,2014,60(5):377-382.
[30]ZIYYAT A,LEFVRE A.Differential gene expression in pre-implantation embryos from mouse oocytes injected with round spermatids or spermatozoa[J].Hum Reprod,2001,16(7):1449-1456.
[31]FERNANDES L T,TOM S A,BENSAID A,et al.Exploratory study on the transcriptional profile of pigs subclinically infected with porcine circovirus type2[J].Anim Biotechnol,2009,20(3):96-109.
[32]LI W T,LIU S Q,WANG Y,et al.Transcription analysis of the porcine alveolar macrophage response to porcine circovirus type 2[J].BMC Genomics,2013,14:353.
[33]FAN H Y,YE Y,LUO Y W,et al.Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus type 2infected PK-15cells[J].J Proteome Res,2012,11(2):995-1008.
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