Transcriptomics-based analysis of the differentially transcribed genes and the mechanism of viral oncogenesis in the lung tumors of sheep naturally infected with Jaagsiekte sheep retrovirus
Ovine pulmonary adenomatosis(OPA)is an infectious lung tumor disease in sheep caused by Jaagsiekte sheep retrovirus(JSRV).To investigate the tumorigenetic mechanism of JSRV in sheep,total RNA was extracted from lung tumor tissues of sheep naturally infected with JSRV(OPA sheep)and lung tissues of healthy sheep.The Illumina HiSeq 4000 high-throughput sequencing platform was used for transcriptome sequencing(RNA-Seq).DESeqR was used to screen the differentially transcribed genes in the lung tissue of healthy and OPA sheep.P<0.05 and log2(Fold change)≥1 were used to screen the significantly differentially transcribed genes.The enrichment analysis of GO function and KEGG signaling pathway of the significantly differentially transcribed genes was performed by GO and KEGG databases,and 10 randomly selected significantly differentially transcribed genes were verified by RT-qPCR.Compared with the control group,1360 up-regulated genes and 783 down-regulated genes were screened,among which 154 significantly up-regulated genes and 212 significantly down-regulated genes.GO functional and KEGG analysis showed that the significantly differentially transcribed genes were significantly enriched in 178 GO terms,including 114 biological process(BP),19 cellular component(CC)and 45 molecular function(MF).They are mainly involved in biological functions such as growth factor activity,post-replication repair,NAD+ diphosphatase activity,nucleoside metabolic process and guanosine nucleotide exchange factor activity and mainly enriched in cell proliferation,differentiation and tumor formation,such as PI3K/Akt,MAPK and Hippo signaling pathways.The results of RT-qPCR were consistent with the results of RNA-Seq screening.In this study,the transcription and expression levels of mammalian Ste20-like protein kinase 1/2(MST1/2),large tumor suppressor 1/2(LATS1/2),Yes-associated protein 1(YAP1)and the phosphorylation level of YAP1(p-YAP1)in the lung tissue samples of sheep in the two groups were detected by RT-qPCR and western blot(WB).Immunohistochemistry(IHC)was used to detect the expression and localization of the core proteins of Hippo signaling pathway in the lung tissue of the two sheep groups.The results of RT-qPCR showed that,compared with the control group,the mRNA transcription levels of MST1 and YAP1 in the lung tissue of OPA sheep were extremely increased(P<0.01,P<0.001),and the mRNA transcription level of LATS1 was significantly increased(P<0.05).WB results showed that MST1/2 had a specific band at 59ku,YAP1 and p-YAP1 had a specific band at 65ku,and LATS1/2 had a specific band at 140ku.Compared with the control group,the expression levels of MST1/2 and p-Yap1 in the lung tissue of sheep in the OPA group were significantly increased(P<0.05),and the expression levels of LATS1/2 and YAP1 were extremely increased(P<0.01).IHC results showed that MST1/2,LATS1/2 and p-YAP1 were located in the cytoplasm of lung tissue in both OPA group and control group.MST1/2,LATS1/2 and p-Yap1 were strongly expressed in lung tissue of OPA group.YAP1 was mainly localized in the cytoplasm of control group,while in the OPA group,YAP1 was mainly localized in the nucleus(with a small amount in the cytoplasm).These results indicated that there were significantly differentially transcribed genes in the lung tissue of sheep between OPA group and control group,which were mainly involved in cell proliferation,tumorigenesis and metastasis,and were mainly enriched in PI3K/Akt,MAPK,Hippo signaling pathways.In particular,YAP,one of the core proteins in the Hippo signaling pathway,may promote the occurrence and development of tumors through nuclear heterotopic.This study provides a new research basis and direction for the tumorigenesis mechanism of JSRV.
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