Identification and validation of novel and annotated LncRNAs in canine B-cell lymphoma by RNA-seq

Introduction. Molecular mechanisms of protein-coding genes, leading to lymphoma development and driving the clinical outcome have partially explained the biology of the canine B-cell lymphoma (cBCL). To expand the knowledge of on noncoding molecules involved in cBCL, we performed an analysis to uncover and characterize both novel unannotated and annotated long non-coding RNAs (lncRNAs). Materials and methods. Using RNA-seq data obtained from cBCLs (50 DLBCLs, 7 FLs and 5 MZLs) and 11 normal lymph nodes (nLNs), we implemented a customized pipeline to detect and GO-functionally enrich novel lncRNAs. Different bioinformatics tools were considered: such as STAR for reads-alignment, StringTie for assembly, CuffCompare to generate the set of lncRNA candidates and, FEELnc to assess the coding-potential score (CPS) were included. Candidates were filtered based on the exon content and differential expression (Limma/EdgeR). Validation through a public available human and cBCL transcriptome datasets (SRA059558) was performed. Results. 1666 novel and 884 already annotated lncRNAs were identified as expressed in the 62 cBCLs and 11 nLNs. Interestingly, a total of 839 novel and 439 annotated lncRNAs were differentially expressed in DLBCL when compared to with nLNs. Only one lncRNA was differently expressed when comparing DLBCL and MZL. To identify a possible mechanism causing this aberrant expression we compared lncRNAs with data obtained from DNA profiling. In DLBCL, the expression of 47 lncRNAs resulted to be correlated to a genomic gain whereas only one lncRNA was associated with genomic loss (FDR<0.05). Deeper investigations including subtyping of DLBCLs, co-expression and survival analysis were also performed. Validation showed that a percent of ~67% and 43% of novel lncRNAs were found in common in the canine or human public datasets, respectively. Conclusions. Our work, so far the most comprehensive analyses for lncRNAs in canine BCL, provides the foundation for future investigations on biological functions and molecular mechanisms of the most significant lncRNAs and their application to the clinic. Acknowledgements. This study was financed by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (Scientific Independence of Young Researchers, SIR, 2014).