Effect Of REV Infection On Posttranscriptional Regulation Of SPF Chicken Embryonic Fibroblasts

Reticuloendotheliosis(RE)is a pathological syndrome caused by reticuloendotheliosis virus(REV)infects avian hosts,including runting syndrome,immunosuppression and tumorigenesis.Since 1958,REV has been widely distributed around the world because of its multiple transmission channels and wide host range.In addition,biologicals such as vaccines may also be contaminated by REV,resulting in vaccination failure,inhibiting the host immune system and reducing its immunity,thus causing co-occurrence of other diseases and causing huge losses to human economy.In this study,we used high-throughput sequencing,real-time fluorescent quantitative PCR and other methods to detect the changes of alternative splicing and miR-155 expression of chicken embryo fibroblasts(CEFs)during the course of REV infection,and explored the regulatory role of CEF and miR-155 in post transcription level of response to REV infection,so as to further study the immunopathogenesis of REV and its immunosuppression and tumor formation in infected animals The study of mechanism provides a new research direction,and provides theoretical basis and new ideas for prevention and treatment of RE.Main results of this study are as follows:(1)Establishment of real-time PCR for detection REVAfter RNA extracted from the replicated virus solution was verified by PCR,bands of 195 bp were observed under ultraviolet lamp,which proved virus solution was REV positive.REV solution was connected with plasmid and diluted ten times continuously.We obtained amplification curve,melting curve and standard curve of REV LTR by real-time PCR.The spacing of recombinant plasmids with different dilutions was uniform when logarithmic growth period was reached,which proved expression difference of recombinant plasmids with different dilutions was linear.At the same time,there was no amplification in negative control,which proved there was no false positive interference in this experiment.Fusion peak of real-time PCR amplification products was single,which proved there was no primer dimer and non-specific amplification.Standard curve of REV LTR is linear regression equation,and selected dilution points are online.Regression equation is y=2.899x+4.464,and correlation coefficient R2=0.9991,which proves reliability is high.(2)Determination of virus titerTCID50 of virus was calculated to be 10-4.625/0.1ml after recording numbers of holes in which cells with CPE for 7 days,that is,when virus was diluted 104.625 times and inoculated with 100 ?l,50% of cells could be pathological.(3)Results and analysis of high-throughput sequencingCEFs were divided into control group C and infection group VB.Each group had three replicates.After high-throughput sequencing,a large number of data were obtained from six samples and the length was 10.2 G-12.92 G,error rate was 0.02%,Q20 was more than 96%,Q30 was more than 90%,and GC content was about 50%.Clean reads still over 94.6% of the original data after removing low quality and contained joints data,which shows the quality of sequencing data is better.After comparing with reference genome,it was found that more than 70% of clean reads could be compared to the reference genome,and uniquely mapped reads was more than 90%.This indicated that the sample was not contaminated,and the selected reference genome was suitable.It was also found that the percentage of protein coding regions that could be compared with the reference genome was all greater than 70%.This indicated that most of the detected genes were protein-coding genes.It also proved that the reads were all from m RNA.A total of 15,973 genes were found in the two comparison groups,of which 6,939 genes had been alternative spliced.Among them,the number of SE events was 16,860,accounting for 89.22% of the total number of events,which means SE is the most common splicing pattern in REV-infected CEFs.SE is also the most important splicing pattern to produce differences that the number of up-regulation events was 183,and the number of down-regulation events was 524.A total of 5,607 genes were found to be differentially expressed in group C and VB,expression of 2,825 genes in group VB was significantly higher than that in control group and expression of 2,782 genes in group VB was significantly lower than that in control group.Differentially expressed alternative splicing genes are mainly related to cellular component in GO enrichment analysis,such as organelle lumen,membrane-bound organelles,cytoplasm and vesicles.Signaling pathways of KEGG enrichment analysis which enriched with differentially expressed alternative splicing genes are phosphatidylinositol signaling pathway,MAPK signaling pathway,apoptosis and focal adhesion.(4)Verification of high-throughput sequencing resultsThe spliced exons of FN1,TNC and SEC61 B were randomly selected and named Fe,Te and Se.As control groups,the three non-spliced exons adjacent to the spliced exons were also detected,named Fn,Tn and Sn.Lanes of Fe,Te and Se had no or only a little PCR products,and the other lanes had obvious and correct bands,indicating alternative splicing events predicted by highthroughput sequencing are real.(5)Relationship between REV infection time and dose and miR-155 expressionAt each time point(12,24,48,72,96 and 120 h)after infection,expression of miR-155 in VB group increased significantly compared with that in C group(P<0.01),and increased with the time of infection.There was a positive correlation between expression of miR-155 and REV infection time,and peaked at 72 h after infection(P<0.001).At the same time,expression of miR-155 also increased with the increase of REV infection titer(P<0.001),and there was a positive correlation between the two.(6)Determination of transfection rate of miR-155 NC,mimics and inhibitorsFluorescence figures were observed under fluorescence inversion microscopy.There was no fluorescence in normal CEFs without transfection,but obvious fluorescence appeared in the cytoplasm of CFFs transfected with miR-155 NC,mimics and inhibitors respectively,and fluorescence efficiency could reach more than 60%.The expression of miR-155 in CFEs transfected with miR-155 mimics increased significantly(P<0.001)and increased gradually with the prolongation of transfection time,reaching a high level at 72 h after transfection and remained basically unchanged,while expression of miR-155 in CFEs transfected with miR-155 inhibitors decreased significantly(P<0.01),it was completely inhibited at 12 h after transfection,and maintained at a low level within 72 h after transfection.There was no significant(P>0.05)difference between non-transfected and miR-155 NC transfected CFEs.(7)Effect of REV infection on viability of CEFsCEFs were divided into C group(control group),V group(REV infection only),VN group(transfected with miR-155 NC after REV infection),VM group(transfected with miR-155 mimics after REV infection)and VI group(transfected with miR-155 inhibitors after REV infection).Compared with group C,viability of group V was significantly(P<0.001)decreased.Compared with group V,viability of group VM increased significantly(P<0.001),while viability of group VI decreased significantly(P<0.01),and there was no significant(P>0.05)difference in cell viability between group VN and group V.(8)Effect of REV infection on apoptosis and related enzyme activity of CEFsResults of flow cytometry showed that the number of apoptotic cells in group V was very significantly(P<0.01)higher than that in group C.Compared with group V,apoptotic rate of group VM was significantly(P<0.01)lower,while apoptotic rate of CEFs was significantly(P<0.05)increased in group VI,and there was no significant(P>0.05)difference in apoptotic rate between group VN and group V.Results of caspase-3 spectrophotometry showed that the production of p NA in group V was significantly(P<0.05)higher than that in group C.Compared with group V,the production of p NA in group VM was decreased,while the production of p NA in group VI was significantly(P<0.01)increased,and there was no significant(P>0.05)difference in the production of p NA between group VN and group V.(9)Effect of REV infection on cell cycle of CEFsResults of flow cytometry showed that compared with group C,number of cells in G0/G1 phase was significantly(P<0.01)increased in group V,number of cells in S phase was significantly(P<0.01)decreased,and number of cells in G2 phase was significantly(P<0.05)decreased.Compared with group V,number of G0/G1 phase cells in group VM decreased significantly(P<0.01),while number of S phase cells increased significantly(P<0.01),number of G2 phase cells also increased;number of G0/G1 phase cells in group VI increased significantly(P<0.01),number of G2 phase cells decreased significantly(P<0.05),and number of S phase cells decreased relatively.In addition,there was no significant difference in cell cycle distribution between group VN and group V.(10)Verification of miR-155 target genesTargetscan,miRanda software and high throughput sequencing results showed that caspase-6 and FOXO3 a were the common target genes of miR-155 in database.Western Blot(WB)detection results showed that caspase-6 and FOXO3 a protein expressions in group V were significantly(P<0.05)higher than those in group C.Compared with group V,caspase-6 and FOXO3 a protein expression in group VM decreased significantly(P<0.05),while FOXO3 a protein expression in group VI increased significantly(P<0.05),and caspase-6 protein expression also increased.In addition,there was no significant(P>0.05)difference in caspase-6 and FOXO3 a protein expression between group VN and group V.These above findings provide an important scientific experimental basis for further elucidate the effect of REV infection on CEFs post-transcriptional regulation,and the important roles of alternative splicing and miR-155 in it.It also provides a new sight for further exploring the pathogenesis of REV in molecular level.