| Chicken Marek’s disease(MD)is a highly infectious lymphoproliferative disease in chickens caused by Marek’s Disease Virus(MDV),which can cause high morbidity and mortality in flocks.One of the major diseases in the healthy development of the industry.Marek’s disease is a viral-induced neoplastic disease that can be prevented by a vaccine.It provides a model for studying diseases of lymphoma formation using natural hosts,and is often used as a model for viral tumor research and treatment at home and abroad.Long-chain non-coding RNA(Lnc RNA)is abnormally expressed in many tumors,which affects the expression of genes involved in tumorigenesis and may promote or inhibit the formation and development of tumors.In this study,MDV BJ-1 strain blood titer titer was detected by plaque test.SPF chicken was challenged with 1000pfu/chicken challenged dose to establish chicken MD tumor model.On 42 d,necropsy challenge group chicken and control group were used.Chicken and record mortality and tumorigenicity.The virus infection was detected by MD agar diffusion test,and the lesions of tissues and organs such as chicken spleen,liver and intestine were observed by necropsy,and the successful construction of MD tumor model was confirmed.Chicken MD splenic tumors and normal spleen tissues were collected and transcriptome sequencing was carried out by RNA-seq sequencing technology.Differentially expressed gene database,differentially expressed Lnc RNA database,signal pathway database and Lnc RNA target gene database were analyzed.The gene co-expression network was constructed and the function of p53-related Lnc RNA was validated in vitro.The results showed that the new Lnc RNA database was used as the data source for analysis,and the new Lnc RNA database records were analyzed with the tools of coding Potential Calculator(CPC),coding Potential Assessment Tool(CPAT),non-coding analysis tool CNCI(Coding-Non-Coding Index),and Pfam protein domain analysis.A comprehensive analysis of coding ability showed that 22181 differentially expressed Lnc RNAs were screened out in MDV infected chickens compared with the control group.The analysis of differentially expressed genes database,signaling pathway database and co-expression database showed that 330 differentially up-regulated signaling pathways were screened(P<0.05),618 differentially up-regulated genes were enriched,of which 91 genes were significantly different(P < 0.01).145 differentially down-regulated signaling pathways(P<0.05)and 392 differentially down-regulated genes were enriched,of which 38 genes were significantly different(P < 0.01).GO functional analysis showed that differentially expressed genes were enriched in 20 items with significant biological functions.Pathway significance enrichment analysis was used to enrich differentially expressed genes in 9 signaling pathways,using KEGG Pathway as a unit.WGCNA(weighted correlation network analysis)was used to construct weighted correlation network.The interaction between Lnc RNA and RNA was analyzed at the expression level.The results showed that 2349 Lnc RNAs predicted the corresponding target genes.To validate the function of TP53BP1-Lnc RNA,a tumor-associated protein p53,in vitro.TP53BP1-Lnc RNA-sh RNA interference vector was constructed and transfected into DF-1 cells by liposome.The transcription level of TP53BP1-Lnc RNA was detected by q PCR and the best interference group was selected.The total protein of DF-1 cells in the best interference group and control group was extracted,and the expression of p53 protein was detected by Western blot.The results showed that the expression level of TP53BP1-Lnc RNA in DF-1 cells transfected with TP53BP1-Lnc RNA-sh RNA-2 interfering vector was significantly decreased(P<0.01);the expression level of TP53BP1-Lnc RNA-sh RNA in DF-1 cells transfected with TP53BP1-Lnc RNA-sh RNA-3and TP53BP1-Lnc RNA-sh RNA-4 interfering vector was significantly decreased(P<0.05).The RNA expression level of p53 gene in DF-1 cells transfected with interfering vector was significantly lower than that in control cells(P<0.05),and the expression level of p53 protein was significantly lower than that in control cells(P<0.05).The results of TP53BP1-Lnc RNA interference validation in vitro showed that the Lnc RNA had an effect on the transcription and protein expression level of p53 gene,which was consistent with the results of RNA-seq.Whether TP53BP1-Lnc RNA had a direct or indirect effect on the expression level of p53 protein needed further study.In this study,high throughput sequencing technology and bioinformatics analysis technology were used to construct the gene transcription level and co-expression network of genes and Lnc RNA in the spleen of chickens infected with MDV.The co-expression network of new Lnc RNAs and differentially expressed genes was obtained,and the TP53BP1-Lnc RNA related to p53 protein expression was verified in vitro.The results of this study will be used to control gene expression in chickens infected with MDV.The molecular mechanism of toxic tumorigenesis can be used as a reference. |
