Interaction between envelope protein of Jaagsiekte Sheep Retrovirus and Hippo signaling pathway is inferred from transcriptome analysis of naturally infected Ovine Pulmonary Adenomatosis

Abstract

Background Ovine pulmonary adenomatosis (OPA) is a contagious lung epithelial tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV), which causes severe economic losses for the sheep industry in the world. The specific oncogenic mechanism of JSRV is not yet clarified. Methods In this study, RNA was extracted from lung tissues of 3 naturally infected OPA cases and 3 healthy individuals for transcriptome sequencing (RNA-Seq). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm the sequencing data. Immunohistochemistry (IHC) and western blot (WB) were performed to confirm the signaling pathway enriched by DEGs that was activated in naturally infected OPA cases. Cell viability, wound-healing, transwell and colony formation assays were performed to assess the cell malignant transformation of sheep trophoblast cells (STCs) transformed with JSRV- env lentivirus in vitro, and then WB was performed to confirm the signaling pathway that had been validated in the lung tissues. Results A total of 366 DEGs (154 up-regulated and 212 down-regulated) were identified by RNA-Seq of lung tissues of naturally infected OPA cases and healthy individuals. GO analysis showed that 366 DEGs were significantly enriched in 178 GO terms, including 114 biological processes, 19 cellular components and 45 molecular functions. KEGG analysis showed that the DEGs mainly enriched in cell proliferation, differentiation, apoptosis and migration, such as PI3K/Akt/mTOR, MAPK and Hippo signaling pathway, and Hippo signaling pathway has never been reported in naturally infected OPA cases. qRT-PCR results of 10 DEGs which were selected randomly were consistent with RNA-Seq results. The protein expression of Hippo signaling pathway were up-regulated in naturally infected OPA lung tissues. Cell viability, wound-healing, transwell and colony formation assays confirmed that JSRV- env lentivirus caused malignant transformation of STCs and JSRV Env increased the protein expression of Hippo signaling pathway. Conclusions This research first identified the changes in the transcriptome level of naturally infected OPA lung tissues. These data confirm that the Hippo signaling pathway is involved in the mechanism of OPA, clarify the interaction between Hippo signaling pathway and JSRV Env, provide further evidence for the tumorigenic mechanism of JSRV.