Interaction Analysis Of MiRNA,mRNA And Prote In Cells Upon Cyprinid Herpesvirus 2

Carassius auratus gibelio breeding industry was heavily affected by Cyprinid herpesvirus 2(CyHV-2).Up to now,the molecular mechanism of the interaction between Carassius auratus gibelio and CyHV-2 were still unknown.Fish cells provide a good platform for the research of fish viruses.The interaction analysis of mi RNA, mRNA and protein will provide a new perspective for further exploring the interaction between virus and host.From the perspective of the host(GiCF),to obtain useful clues for the immun-regulation of CyHV-2 infection and pathogenesis,this paper explored the differential expression of miRNA, mRNA,and protein of GiCF at different time when CyHV-2 infected GiCF.Based on the above statements,the research results have been obtained are as follows:(1)Stability analysis of reference genesIn this experiment,we screened out the reference genes of the Carassius auratus gibelio,which provided a guarantee for the accuracy of the high-throughput sequencing results verification in the next chapter.The reference gene was theoretically maintained at a relatively stable level under different conditions.However,genes that are stably expressed are almost non-existent underlying different conditions.Therefore,in order to improve the accuracy of quantitative PCR results,it was necessary to screen the reference genes under the different processing conditions.The online software ge Norm,Norm Finder,Best Keeper,and Delta Ct were combined with analysis of Ct value and ge Norm stability value.The results showed that β-actin and EF-1α can be used as reference genes in different tissues of healthy crossbred Carassius auratus gibelio;When CyHV-2 infected kidney and GiCF at different times,β-actin can be used as reference gene;When CyHV-2 infected spleen tissue at different times,EF-1α had the best stability.The expression analysis of PIN1 at different time of infection in the kidney tissues of Carassius auratus gibelio further confirmed the applicability of its reference gene.(2)Combine analysis of miRNA and mRNAUsing the Illuminc Hiscq 4000 sequencing platform,control groups(A1,A2,A3),48 h infection(B1,B2,B3)and 96 h infection(C1,C2,C3)GiCF cells were constructed with 9 mRNA libraries and 9 s RNA library.A total of 781,844,440 Clean reads were obtained from the nine mRNA libraries.A total of 292,228 unigenes with an average length of 736.65 were obtained by Trinity splicing.A total of 27,014,779 Clean reads were obtained from the nines RNA libraries.Compared with the miRNAs of all animals in miRNA Base,a total of 1046 mature miRNAs were identified(409 Known miRNAs and 631 Novel miRNAs).Among of these Known miRNAs,28 miRNAs participate in 20 immune-related pathways.Compared with the miRNA sequencing results of kidney tissues of Carassius auratus gibelio found in 2018 by Lu Jianfei,7 miRNAs have significant differences both in vivo and in vitro(refer to Lu Jianfei’s article for setting differential screening conditions,P <0.01).To obtain miRNAs with significant differences as much as possible,we set more precise differential screening conditions: | log2 FC |> 1,TPM> 10,P <0.01.Differential expression analysis was performed on the Known miRNAs.7 miRNAs with significantly differentially expression(5 down-regulated and 2 up-regulated)were finally screened and verified by RT-qPCR,and the interaction network diagram of miRNA and its targeted mRNA was drawn.6 potential mRNA targets which involved in immune-related pathways were verified.Target prediction and functional analysis of these regulated miRNAs indicate differentially expressed miRNAs at the host(GiCF cell)level participate in the PPAR signaling pathway,p53 signaling pathway,and Fox O signaling pathway underlying the influence of CyHV-2 infection.(3)Combine analysis of mRNA transcriptome and proteomeTo further explore the molecular mechanism of CyHV-2 infected Carassius auratus gibelio and the corresponding immune mechanism of the host.In this chapter,transcriptome sequencing technology and TMT marker quantitative proteomics technology were used to screen differentially expressed genes and proteins of GiCF cells after CyHV-2 infects GiCF cells 48 h and 96 h.The results showed that: compared with the control group,there are 11335 differentially expressed mRNAs after CyHV-2 infected GiCF cells 48 h,and there are 19421 differentially expressed mRNAs after CyHV-2 infected GiCF cells 96 h.Proteomic analysis revealed that a total of 9008 proteins were identified.Compared with the control group,there are 169 differential proteins after CyHV-2 infected GiCF cells at 48 h,and there are 502 differential proteins after CyHV-2 infected GiCF cells at 96 h.The results of combined transcriptome and proteome analysis showed that there were 10 and 158 differentially co-expressed genes(c DEGs)in CyHV-2 infected GiCF cells at 48 and 96 h,compared with the control group,respectively.Among of them,the differential co-expression of GNG7 and Casease8 were significantly up-regulated,and the differential co-expression of HSP90 A,CD140,RRM2,THBS1,BTF3,RAB7 A,as well as CNTFR were significantly down-regulated,which indicated that PI3k-AKT,Apoptosis and Metabolic pathways were activated after CyHV-2 infection,but Phagosome and JAK-STAT signaling pathway was suppressed.The qPCR results further showed that the related genes in these signaling pathways had different trends consistent with the proteome changes at different time points after CyHV-2 infection.