Identification and Quantification of Novel RNA Isoforms in Horn Cancer of Bos indicus by Comprehensive RNA-Seq

Horn cancer (HC) is a squamous cell carcinoma of horn, commonly observed in Bos indicus of the Asian countries. To elucidate the complexity of alternative splicing present in the horn cancer, high throughput sequencing and analysis of HC and matching normal horn tissue was carried out. A total of 535,067 and 849,077 reads were analyzed after stringent quality filtering for HN and HC, respectively. We found 13,020 and 12,728 known isoforms were detected in HC and horn normal. Additionally, 130 gene isoforms were found to be differentially expressed between both the conditions. We also identified 4,786 novel splice variants comprising 2432, 2055 and 298 splice variants present uniquely in HC, uniquely in horn normal and in both condition, respectively. Pathway analysis and gene ontology of isoforms with differential expression revealed deregulation of cancer pathways, angiogenesis, epithelial cell differentiation and cell death. Based on pathway clustering and in-silico verification, selected 102 novel splice variants were further analysed with respect to change in protein sequence. Blastp of translated protein sequences revealed insertion/deletion of amino acids, premature stop codon and loss of functional domains. Finally, we selected and confirmed expression of 14 novel splicing events by RT-qPCR. Future studies targeted at in depth characterization of these potential candidate splice variants might be helpful in development of relevant biomarkers for early diagnosis of HC. We have confirmed presence and expression of selected splice variants in additional horn cancer and normal samples through quantitative real time PCR. As, these variations are not yet characterized in systemic way in such a detail, findings of this study provides important insights and serves as a milestone for further characterization of these variations and their association with the horn cancer.