Influence Of The Expression Of MiRNA And Its Target Gene MRNA In SPF Chickens Infected By Reticuloendotheliosis Virus

Reticuloendotheliosis(RE)is an infectious,tumorigenic disease caused by avian reticuloendotheliosis virus(REVs).REV mainly causes the growth retardation syndrome,immunosuppression and the symptoms of chronic tumor in sick birds.Since REVs was first isolated in 1958,it has been reported in many countries and regions in the world.In 1986,Chinese scholars were first isolated REV in Nanjing.The chicken,turkey,duck,peacock,quail,partridge,pheasant and guinea fowl can be infected by REVs.The most susceptible animal is turkey.Chicken is the main research object of REVs infection in the laboratory.The main transmission mode of REV is horizontal communication.In addition,the transmission mode of REV also includes vertical transmission,insect transmission,environmental pollutants transmission and so on.The spread of the virus will cause a variety of virus vaccine immune failure and reduce the immune function of animals.It makes that it is easier for other viruses to infect animals.It can cause many kinds of virus mixed infection,disease of chicken deaths and huge losses in poultry industry.The REV infection model chickens were used in this study.We successfully established animal model of SPF chickens infected with reticuloendotheliosis virus.The dynamic changes of the differential expression of mi RNA and its target gene mRNA expression in the bursa of Fabricius in REV infected SPF chickens were detected by high throughput sequencing and real-time fluorescent quantitative PCR.Combined with REV infection of SPF chickens clinical symptoms,we investigated the dynamic changes of the above indexes of SPF chickens infected with REV and their effects on apoptosis and tumorigenesis.The aim of this study was to investigate the effect of REV infection on mi RNA expression in SPF chickens infected with REV,and to study the effect of REV on the expression of apoptosis and tumor related genes:1.Establishment of REV infected SPF chicken disease modelIn this study,we make the 1 day old SPF chickens infected with REV by intraperitoneal injection of the virus.We judged whether the chickens were infected with REV successfully by the analysis of the clinical symptoms,the observation of the pathological changes and the PCR detection.The results showed that compared with the control chickens,the experimental chicken have typical symptoms of growth arrest syndrome which are mental depression,rare and untidy feathers,decreased food intake and reduced weight after intraperitoneal injection of REV.Throughthe experiment chickens were dissected,we can found that the volume of the immune organs such as bursa of Fabricius and thymus was significantly reduced.PCR was used to detect the bursa of Fabricius in experimental chickens.The results showed that the specific conservative sequence REV of LTR was successfully amplified in the chicken bursa of Fabricius.However,the same day old chickens did not amplify the gene.All of the above indicators have been proved that RE chickens can be successfully replicated by injecting REV solution into the SFP chickens.2.Establishment and application of real time fluorescent quantitative PCR for detection of REVSo far,many methods have been reported for the detection of REV,including common PCR,real-time fluorescent quantitative PCR(RT q-PCR),enzyme-linked immunosorbent assay(ELISA),indirect immunofluorescence(IF),loop mediated isothermal amplification(LAMP)and so on.These methods have their own advantages,but also have shortcomings.In this study,we established a quantitative PCR detection method based on the LTR gene conserved sequence of REV in recent years.Through the optimization found that its optimal annealing temperature is57 ?,the correlation coefficient is R2=0.996753,the regression equation: y=32.311-3.142 x.By using the real-time fluorescent quantitative PCR method,we detected the contents of virus in thymus,spleen and bursa of Fabricius in the SPF chickens infected with REV for twenty-first and twenty-eighth day.It was found that the LTR gene was detected in the thymus,spleen and bursa of Fabricius of chickens on the twenty-first and twenty-eighth day after REV infection.The content of virus in bursa of Fabricius was significantly higher than that of thymus and spleen,but there was no statistical difference in thymus and spleen.3.High throughput assay of mi RNA in bursa of Fabricius cells of REV infected chickensIn this study,we detected the expression of mi RNA in bursa of Fabricius in REV infected chickens by high-throughput assay on the twenty-first and twenty-eighth day after REV infection.Through the analysis of data quality,it was found that the sequence length of each group was 0.597G-0.776 G.The error rate was 0.01%.The Q20 of the data in the four groups were higher than98% and the Q30 was higher than 97%,which were higher than the industry standard.The content of GC was about 50%,which would not affect the sequencing.The sequencing data were filtered to remove the data of the genome,which affected the quality of the data.The clean reads was more than 85% of the total reads.Mapping to the reference genome clean reads accounted for more than82% of the total reads,the above results show that the sequencing data quality is good and high reliability.We found that the expression of 63 mi RNAs was significantly changed on the twenty-first day after SPF chickens infected with REV,which has 30 up-regulated and 33down-regulated mi RNA;the expression of 25 mi RNAs was significantly changed on the twenty-eighth day after SPF chickens infected with REV,which included 8 up-regulated,17down-regulation mi RNA by differential expression analysis of clean reads.4.Verification the results of high-throughput sequencingThe real time fluorescent quantitative PCR method was used to validate the results ofhigh-throughput sequencing.The 10 up-regulated and down-regulated differentially expressed mi RNA were random Ly selected from results of the analysison on the twenty-first day and twenty-eighth day after SPF chickens infected with REV,respectively.And their expression was detected.Through the correlation analysis,we found that the detection results of 10 mi RNAs expression by high-throughput sequencing was positively correlated with the detection results of10 mi RNAs expression by real-time fluorescent quantitative PCR on the twenty-first and twenty-eighth day after REV infection.The correlation coefficients were: R2=0.9633 and 0.9796,respectively.The above results showed that high throughput sequencing results were accurate.5.Research on the mechanism of apoptosis induced by REV infectionThe apoptosis of bursa of Fabricius in SPF chickens was obvious on the twenty-first day after REV infection.We found that the expression of mi RNA target gene Caspase-6,Caspase-7 and P27 were significantly increased,while the expression of Mcl-1 was significantly decreased.Caspase-6and Caspase-7 are considered to be the key factor of cell apoptosis and tumor suppressor protein P27 can promote apoptosis.They are both pro apoptotic factor.The increase in their expression suggests that the apoptotic process is promoted.The Mcl-1which is Bcl family member has the function of inhibiting apoptosis,and it is a typical inhibitor of apoptosis factor.the decrease of Mcl-1 expression also indicates that apoptosis is promoted.By analyzing the negative correlation between target gene and its regulation of mi RNA expression,we found that there was a significant negative correlation,and the negative correlation coefficient was R2=0.9533,which indicated that mi RNA could regulate the target gene.These results indicate that REV can promote the expression of pro apoptotic factor mRNA,and inhibit the expression of apoptosis inhibiting factor,which can induce the apoptosis of bursa of Fabricius cells by regulating the expression of mi RNA.6.Effects of REV infection on tumor associated target genesThe expression of target gene CCNA1,CCNB2,CCND3 and c-myc of the differentially expressed mi RNA were detected to varying degrees up-regulation on the twenty-eighth day after REV infection.CCNA1,CCNB2 and CCND3 are different isoforms of cyclin,which play a role in promoting apoptosis,and had a higher expression in the tumor cells.C-myc is a proto oncogene,and its high expression can prove the occurrence or obvious trend of tumor.By analyzing the negative correlation between these genes and their mi RNA,we found that the negative correlation coefficient was R2=0.9533,which indicated that they had significant negative correlation.These results suggest that REV promotes the development of cancer by promoting the expression of cyclin and activating proto oncogenes.