Role of microRNAs in Jaagsiekte sheep retrovirus infection

Abstract

Ovine pulmonary adenocarcinoma (OPA) is a lung cancer that affects sheep, caused by jaagsiekte sheep retrovirus (JSRV). OPA is present in most sheeprearing countries of the world, but, at present, there are no reliable early-stage tests to diagnose the disease, and OPA continues to pose an animal welfare threat and cause substantial economic losses. In addition, OPA is a valuable animal model to study early oncogenic events in human lung cancer. Specifically, OPA and some types of human lung cancer present similarities in activated signalling pathways (Ras-MEK-ERK1/2 and PI3K-AKT-mTOR) and their association with type II pneumocytes. Nevertheless, study of the molecular pathogenesis of OPA has been hindered due to the lack of a permissive cell line for JSRV replication. JSRV encodes an unusual envelope protein (Env) which is actively oncogenic and sufficient to drive transformation in vivo and in vitro. Despite the lack of a permissive cell line, early oncogenic events induced by JSRV can be studied by transfection of cell lines with plasmids encoding JSRV Env. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and essential cell processes such as cell proliferation and apoptosis. miRNAs are being extensively studied as biomarkers of several diseases, including cancer. The aims of this project were to investigate the role of miRNAs in the early oncogenic events induced by JSRV and to investigate their potential as OPA biomarkers. miRNA expression levels were investigated using small RNA sequencing in lung tissue from cases of experimentally induced OPA. No evidence of JSRV-encoded miRNAs was found, but levels of 40 miRNAs were found differentially expressed between affected and control sheep. Of those, upregulation of nine microRNAs (miR-135b, miR-182, miR-183, miR-21, miR-200b, miR-205, miR-31, miR-503 and miR-96) was confirmed by RT-qPCR in experimental and natural cases of OPA, suggesting that increased levels of these miRNAs were characteristic of OPA affected lung tissue. To investigate miRNAs as potential biomarkers, miRNA expression was measured in serum and bronchoalveolar lavage fluid (BALF) macrophages of OPA affected sheep. small RNA sequencing revealed 74 microRNAs and 85 miRNAs differentially expressed in serum and BALF macrophages, respectively. Interestingly, BALF macrophage microRNA expression was found to resemble more closely that of OPA affected sheep lungs. In addition, miRNA expression levels varied at different stages of the disease and no miRNAs were found to be consistently dysregulated in serum of OPA affected animals. Discordances in miRNA signatures in lung tissue and serum are not entirely unexpected. Lung tissue miRNAs might represent the tumour microenvironment and localised response to it, whereas miRNAs in serum may represent the global state of the animal, and tumour miRNAs might be released into circulation at low levels, making them difficult to detect. Expression of the nine upregulated miRNAs was then investigated in in vitro models to study their involvement in transformation. Lentiviral vectors encoding green fluorescent protein (GFP) or a GFP-2A-Env fusion protein were produced and used to transduce cell lines. Transformation was verified by immunocytochemical detection of the transformation markers P-Akt and PERK1/2. Nevertheless, miRNA expression levels in culture did not resemble those observed in lung tissue of OPA-affected sheep. These differences might be due to species variation, upregulation of miRNAs late in the transformation process, or involvement of other cell types in tissue besides the transformed cells. To study these questions further, JSRV and the GFP-2A-Env encoding lentiviral vectors were used to infect lung slices in culture. Expression levels of miRNAs did not, in any of the cases, resemble lung tissue findings. Fewer than 5% of cells in lung slices were found to be infected, suggesting that changes in miRNA expression could be masked by the background of normal cells. Nevertheless, increasing JSRV21 concentration did not yield higher infection levels, indicating that those might be more dependent on the availability of JSRV’s target cells, dividing type II pneumocytes, than viral concentration. Taken together, this study has revealed new information on miRNA expression in OPA-affected sheep, including expression patterns in lung and serum. Future work should focus on developing a permissive replication system to allow the study of miRNAs in early JSRV-induced transformation events.