Dissecting the pathogenesis of ovine pulmonary adenocarcinoma with RNA-Seq
Lead Research Organisation:
University of Edinburgh
Department Name: The Roslin Institute
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
Ovine pulmonary adenocarcinoma (OPA) caused by jaagsiekte sheep retrovirus (JSRV) is an important disease of sheep that causes significant losses to the sheep industry and is an important animal welfare issue. Our aim in this project is to dissect the molecular nature of pathogenesis of OPA using high-throughput transcriptome sequencing (RNA-Seq) and to examine the function of selected dysregulated pathways in an ovine lung culture system.
We will perform RNA-Seq and microRNA-Seq on tumour cells and infiltrating macrophages from microdissected tumour tissue from experimentally infected lambs. We will also obtain sequence from epithelial cells and macrophages from uninfected control lung. Differentially regulated genes and gene networks that are likely to be important in pathogenesis will be identified. Preliminary data has confirmed that this approach can identify differentially regulated genes in these tissues. Next, we will determine the host transcriptional response to infection by a non-transforming JSRV mutant. This will allow differentiation of host gene expression pathways that are altered in response to infection alone from those altered due to tumour growth and will determine whether oncogenesis has a role in the lack of adaptive immunity to JSRV in sheep.
The differential expression of selected target genes will be validated on a panel of natural and experimental cases of OPA using RT-qPCR and immunohistochemistry. Subsequently, we will test the function of selected pathways in JSRV pathogenesis in vitro using an ovine lung tissue culture system. This system allows the manipulation of the expression of genes of interest to predict and directly test the function of selected targets in vitro. Collectively, these objectives will allow us to define the molecular events occurring during JSRV pathogenesis. This information will underpin the design of new control strategies for OPA, e.g., by identifying new diagnostic targets and vaccination strategies.
We will perform RNA-Seq and microRNA-Seq on tumour cells and infiltrating macrophages from microdissected tumour tissue from experimentally infected lambs. We will also obtain sequence from epithelial cells and macrophages from uninfected control lung. Differentially regulated genes and gene networks that are likely to be important in pathogenesis will be identified. Preliminary data has confirmed that this approach can identify differentially regulated genes in these tissues. Next, we will determine the host transcriptional response to infection by a non-transforming JSRV mutant. This will allow differentiation of host gene expression pathways that are altered in response to infection alone from those altered due to tumour growth and will determine whether oncogenesis has a role in the lack of adaptive immunity to JSRV in sheep.
The differential expression of selected target genes will be validated on a panel of natural and experimental cases of OPA using RT-qPCR and immunohistochemistry. Subsequently, we will test the function of selected pathways in JSRV pathogenesis in vitro using an ovine lung tissue culture system. This system allows the manipulation of the expression of genes of interest to predict and directly test the function of selected targets in vitro. Collectively, these objectives will allow us to define the molecular events occurring during JSRV pathogenesis. This information will underpin the design of new control strategies for OPA, e.g., by identifying new diagnostic targets and vaccination strategies.
Planned Impact
Ovine pulmonary adenocarcinoma (OPA, also known as jaagsiekte) is a common infectious disease of sheep that is
widespread in the UK and in many countries where sheep are farmed. OPA causes significant economic losses to
producers and the severe respiratory distress it produces is a serious animal welfare concern. Our aim in this project is to
dissect the molecular nature of pathogenesis of OPA using state of the art genetic technologies. Our vision is that the
outputs of this research will stimulate new avenues of research in the area of pulmonary biology and deliver information
that will underpin the design of novel strategies to control OPA.
WHO WILL BENEFIT FROM THIS RESEARCH?
A variety of stakeholder groups will be beneficiaries of the proposed research in the UK and elsewhere, including research
scientists, academic and government research organisations, farmers, veterinarians, government and the public.
HOW WILL THEY BENEFIT?
Research scientists and research organisations will benefit from the increased understanding of gene expression and lung
function in sheep. While this has obvious impact for researchers studying ruminant disease, it will also have much broader
relevance for anyone studying pulmonary biology or pathology. The knowledge gained will support research into other lung
diseases, including infectious disease, inflammatory disease and other disorders of immune function. We also expect that
the genetic information generated in this project will provide the intellectual framework for greater exploitation of sheep as
an improved animal model of human pulmonary biology. The identification of pathways activated during oncogenesis in
OPA will be valuable for scientists studying human lung cancer or other epithelial tumours, while tumour biomarkers
identified in OPA could have utility in diagnosing in human lung cancer. Furthermore, the use of RNA-Seq in sheep will
demonstrate the utility of this technology for investigating other species with relatively uncharacterised genomes. This will
also support the study of related species that are not target species for genome sequencing.
Sheep farmers and associated veterinary industries will benefit from the increased understanding of the pathogenesis of
OPA because this information is required to design novel strategies for controlling this disease. The cost of OPA to
individual farmers can be significant, particularly if breeding rams are affected, and the disease can have disastrous effects
on the financial sustainability of sheep farming in some geographical areas, with consequent negative effects on associated
industries and local communities. More broadly, respiratory disease in ruminant livestock species has enormous economic
impact and the detailed characterisation of lung function at the transcriptome level, including microRNAs, will also
contribute to knowledge underpinning efforts to control those diseases.
Government and the wider public will benefit from this research in a number of ways. Improved animal health and welfare
will result in more efficient animal production, contributing to greater food security and safety. In addition, improving
efficiency in farming reduces its impact on the environment, leading to reduced usage of natural resources and reduced
generation of greenhouse gases. Furthermore, the enhanced financial security of sheep producers will help to improve the
sustainability of rural communities. While this has obvious advantages for those living in those communities, it also helps to
sustain an inviting rural landscape that supports wider industries such as tourism and leisure.
widespread in the UK and in many countries where sheep are farmed. OPA causes significant economic losses to
producers and the severe respiratory distress it produces is a serious animal welfare concern. Our aim in this project is to
dissect the molecular nature of pathogenesis of OPA using state of the art genetic technologies. Our vision is that the
outputs of this research will stimulate new avenues of research in the area of pulmonary biology and deliver information
that will underpin the design of novel strategies to control OPA.
WHO WILL BENEFIT FROM THIS RESEARCH?
A variety of stakeholder groups will be beneficiaries of the proposed research in the UK and elsewhere, including research
scientists, academic and government research organisations, farmers, veterinarians, government and the public.
HOW WILL THEY BENEFIT?
Research scientists and research organisations will benefit from the increased understanding of gene expression and lung
function in sheep. While this has obvious impact for researchers studying ruminant disease, it will also have much broader
relevance for anyone studying pulmonary biology or pathology. The knowledge gained will support research into other lung
diseases, including infectious disease, inflammatory disease and other disorders of immune function. We also expect that
the genetic information generated in this project will provide the intellectual framework for greater exploitation of sheep as
an improved animal model of human pulmonary biology. The identification of pathways activated during oncogenesis in
OPA will be valuable for scientists studying human lung cancer or other epithelial tumours, while tumour biomarkers
identified in OPA could have utility in diagnosing in human lung cancer. Furthermore, the use of RNA-Seq in sheep will
demonstrate the utility of this technology for investigating other species with relatively uncharacterised genomes. This will
also support the study of related species that are not target species for genome sequencing.
Sheep farmers and associated veterinary industries will benefit from the increased understanding of the pathogenesis of
OPA because this information is required to design novel strategies for controlling this disease. The cost of OPA to
individual farmers can be significant, particularly if breeding rams are affected, and the disease can have disastrous effects
on the financial sustainability of sheep farming in some geographical areas, with consequent negative effects on associated
industries and local communities. More broadly, respiratory disease in ruminant livestock species has enormous economic
impact and the detailed characterisation of lung function at the transcriptome level, including microRNAs, will also
contribute to knowledge underpinning efforts to control those diseases.
Government and the wider public will benefit from this research in a number of ways. Improved animal health and welfare
will result in more efficient animal production, contributing to greater food security and safety. In addition, improving
efficiency in farming reduces its impact on the environment, leading to reduced usage of natural resources and reduced
generation of greenhouse gases. Furthermore, the enhanced financial security of sheep producers will help to improve the
sustainability of rural communities. While this has obvious advantages for those living in those communities, it also helps to
sustain an inviting rural landscape that supports wider industries such as tourism and leisure.
People |
ORCID iD |
| Michael Watson (Principal Investigator) |
Publications
Karagianni AE
(2019)
Transcriptional Response of Ovine Lung to Infection with Jaagsiekte Sheep Retrovirus.
in Journal of virology
| Description | 1. Whole transcriptome profiling in JSRV infected lung samples of sheep (RNAseq and microRNA) There were four JSRV infected sheep and four uninfected specific pathogen-free (SPF) lambs used to collect samples. Uninfected lung tissue came from age and sex-matched control lambs. There were 3 males and 1 female animals which were culled after 66, 71, 85 and 85 days old respectively. The RNA-seq data of these samples revealed the expression profile in all infected samples. The pipeline for RNA-seq data analysis consist the following steps - Tophat -> HTSeq-counts -> edgeR -> Pathway analysis. Differentially expressed genes (FDR < 0.05) were listed. The infected samples have the diverse virus counts and the normal tissue contamination during sampling, so the normalization was stimulating practice. The virus counts were also imported with sheep genes counts in edgeR package so that the genes expression can be normalized using relevant virus expression and the list of differentially expressed genes could be listed. The fold-changes of significantly expressed genes were underestimated because of predisposition of gene expression by normal tissues present in infected samples, specifically those genes which are down regulated by giving us the correct order of differentially expressed genes. Second objective of this study was comparing the significantly expressed genes in JSRV infected sheep samples to the published data on human NSCLC studies, with a particular focus on LUAD (lung adenocarcinoma) and LUSC (squamous cell lung carcinoma) samples. The quantification of gene expression by HTSeq-counts from RNA-Seq data for LUAD and LUSC are publicly available on GDC portal (portal.gdc.cancer.gov). There were 57 LUAD and 49 LUSC patients selected where we had corresponding normal and tumor both data available. The HTSeq counts of human genes were analysed using edgeR pipeline same as used in sheep data and the differentially expressed genes were listed in different groups. The list of differentially expressed genes in human data were filtered for only those which have orthologous gene in sheep. The comparison of expression of these short-listed genes was made using the correlation between expression profile in human and in sheep. This analysis provides us with important information regarding the pathogenesis of OPA at a transcriptional level and reveals new insights into the use of sheep as an animal model for human lung adenocarcinoma. The microRNA sequencing was also done on the same samples. This data were analysed to find the differentially expressed microRNA in infected samples, or possibly any virus microRNA. The raw data were trimmed to remove adaptors. The trimmed reads were then filtered by expected microRNA length (17-25bp). The selected reads then mapped on miRBase database using Novoalign. The sam file was then analysed to find out the expressed microRNA in the following priority order - sheep, bovine, human and then the rest of microRNAs. The count file was made and imported into edgeR to list the fold change and fold-discovery rate. The differentially expressed microRNAs were listed using FDR < 0.05. 2. RNASeq and microRNA data of alveolar macrophages in lung of sheep There were alveolar macrophages of 5 lung samples of Ovine pulmonary adenocarcinoma (OPA) infected sheep, 3 healthy sheep lung samples and 3 lung samples of parasite infected sheep taken for RNA-Seq and micro-RNA sequencing. The RNA-seq data were analysed using Kallisto pipeline. The reads were mapped on sheep transcriptome using Kallisto. The pseudocounts for each transcript were imported in DESeq using Tx-import and analysed to find differentially expressed genes in all experimental groups. The list of significant genes expressed in OPA samples compared to healthy samples was then analysed for important pathway analysis. It was interesting finding from these data was the expression profile between healthy and parasite infected sheep samples were similar. There were many differentially expressed genes present in parasite infected samples compared to healthy. The microRNA sequencing was also done on the same samples. This data were analysed to find the differentially expressed microRNA in infected samples, or possibly any virus microRNA. The raw data were trimmed to remove adaptors. The trimmed reads were then filtered by expected microRNA length (17-25bp). The selected reads then mapped on miRBase database using Novoalign. The sam file was then analysed to find out the expressed microRNA in the following priority order - sheep, bovine, human and then the rest of microRNAs. The count file was made and imported into edgeR to list the fold change and fold-discovery rate. The differentially expressed microRNAs were listed using FDR < 0.05. The list of targeted genes for each significant microRNA was downloaded from TargetScan. The fold-change of these target genes were listed from RNA-seq results and plotted to see if there is any significant expression in these target genes. 3. RNASeq and microRNA data of laser microdissected OPA tumour There were 3 OPA infected lambs, 3 mock infected lambs (i.e. no virus) and 3 mutant-infected lambs which was a non-transforming mutant virus. There were some issues regarding the low amount of RNA to be sequenced which lead to detailed analysis of data. The abundance of RNA-seq data in healthy samples were quite higher than the other sample. To achieve the comparable size of data, the healthy sample data were sub-sized using Seqtk. The reads were then mapped on transcriptome using Kallisto and counts were obtained. The counts were imported in DESeq using Tx-import and analysed to find differentially expressed genes in all experimental groups. Only 4%, 10% and 12% of reads of healthy data were mapped on transcriptome which leaves the rest of data in query (52% was the average mapping for othere samples). Reads were then trimmed again using different tools like cutadapt, trimmomatic, trimgalore etc. The filtered data then mapped on reference genome of Sheep and virus using hisat2 which had 46%, 28% and 21% fata mapping on reference. These figures are still lower than other samples which have 80% average data mapping on reference. The data was then also mapped on RNAcentral database assuming non-coding RNA contamination but this was not the case. This is still under investigation. The microRNA sequencing was also done on the same samples. The abundance of data in all samples were comparable. This data were analysed to find the differentially expressed microRNA in infected samples, or possibly any virus microRNA. The raw data were trimmed to remove adaptors. The trimmed reads were then filtered by expected microRNA length (17-25bp). The selected reads then mapped on miRBase database using Novoalign. The sam file was then analysed to find out the expressed microRNA in the following priority order - sheep, bovine, human and then the rest of microRNAs. The count file was made and imported into edgeR to list the fold change and fold-discovery rate. 4. miRNA data of sheep serum There were 3 serum samples from JSRV infected sheep and 3 serum samples from healthy sheep were sequenced for micro-RNA. This data were analysed to find the differentially expressed microRNA in infected samples, or possibly any virus microRNA. The raw data were trimmed to remove adaptors. The trimmed reads were then filtered by expected microRNA length (17-25bp). The selected reads then mapped on miRBase database using Novoalign. The sam file was then analysed to find out the expressed microRNA in the following priority order - sheep, bovine, human and then the rest of microRNAs. The count file was made and imported into edgeR to list the fold change and fold-discovery rate. While selecting sample based on length, there was a noticeable peak at the length between 28bp and 32bp. To determine the type of these sequences, there were mapped on RNAcentral and it showed most of these sequences are non-coding RNA such as t-RNA, piRNA, precursor-RNA, lncRNA etc. 5. miRNA data of sheep serum of lamb and adult sheep There were 5 different groups of sheep sampled using serum for microRNA sequencing. Each group has 6 samples and groups are natural late OPA infected adult sheep, natural early OPA infected adult sheep, healthy adult, experimental OPA infected lamb and healthy lamb. The raw data were trimmed to remove adaptors. The trimmed reads were then filtered by expected microRNA length (17-25bp). The selected reads then mapped on miRBase database using Novoalign. The sam file was then analysed to find out the expressed microRNA in the following priority order - sheep, bovine, human and then the rest of microRNAs. The count file was made and imported into edgeR to list the fold change and fold-discovery rate. All groups were compared to each-other for significant expression of microRNA. The results were suggested that the number of mismatched allowed while analyzing mapping output need to be reduced to remove the micro-RNAs found as some kind of contamination. |
| Exploitation Route | We intend to define the mechanism of action of OPA which will be of interest to those combating the disease (vets/researchers/vaccine developers) |
| Sectors | Agriculture, Food and Drink,Healthcare |
| Description | Understanding how JRSV infects and causes disease is of huge importance for the future design of intervention strategies such as vaccines and drugs. Our findings will provide such fundamental understanding. |
| First Year Of Impact | 2016 |
| Sector | Agriculture, Food and Drink |
| Impact Types | Economic |
| Description | David Griffiths, Moredun |
| Organisation | Moredun Research Institute |
| Country | United Kingdom |
| Sector | Charity/Non Profit |
| PI Contribution | We have been working with David Griffiths at The Moredun Research Institute since 2011 in the study of ovine pulmonary adenocarcinoma |
| Collaborator Contribution | David Griffiths is an expert in the study of ovine pulmondary adenocarcinoma, how the virus functions and how we might design intervention strategies such as vaccines. My group is expert in the use of genomics and bioinformatics and together we are using sequencing to understand how the virus interacts with its host |
| Impact | NA |
| Start Year | 2011 |