Long non-coding RNAs (lncRNA) regulate gene transcription and diverse cellular functions. We have recently reported a core inflammatory and metabolic ileal gene signature in treatment naïve pediatric Crohn Disease (CD), but the potential role of lncRNA was not known. We hypothesized that differences in ileal lncRNA would be associated with differences in protein-coding gene expression, suggesting a role for lncRNA in regulating those gene signatures.Methods:
Poly-A selected RNA-seq and Ensembl annotation were used to define differentially expressed ileal genes with false discovery rate correction of 0.05 and fold change difference of 1.5 between treatment naïve pediatric CD (n = 139) and controls (Ctl, n = 42). “Guilt-by-association” analyses identified potential lncRNA function by testing for co-expression of lncRNA with protein-coding mRNA. RT-PCR was used to test lncRNAs regulation by IL-1β in Caco-2 enterocytes model.Results:
We identified 2043 protein-coding genes and 546 lncRNA differentially expressed between CD and Ctl. Unsupervised hierarchical clustering using only the 546 lncRNA showed that all Ctl grouped in cluster one and most of the CD patients grouped in cluster 2, similar to clustering using just the 2043 coding genes. Supervised Support Vector Machine classification was used to develop a model utilizing a training sub-set of patients and the 2043 protein-coding or 546 lncRNA genes. Applying the model to an independent validation sub-set of patients resulted in accurate classification of 86/89 (97%) using the 546 lncRNA and 87/89 (98%) using the 2043 protein coding-genes. CDKN2B-AS1 (ANRIL) lncRNA, which has previously been shown to regulate gene transcription in-cis (in vicinity) and in-trans (at distant loci) in carcinogenesis and cardiovascular disease, was within the top 10 down-regulated genes (FC > 6) in CD and showed co-expression (Pearson correlation [r > 0.75]) with 365 genes. Functional annotation enrichment analyses of these 365 suppressed genes showed enrichment for entities associated with organic acid (P < 6.01E-20) and lipid metabolic processes (P < 5.47E-16). LINC01272 lncRNA, which was previously shown to be up-regulated in the colon of adult IBD patients, was within the top 5 up-regulated genes (FC > 9) and showed co-expression with 116 genes. Functional annotation enrichment analyses of these 116 genes showed enrichment for entities associated with inflammatory response (P < 8.08E-27), response to bacterium (P < 2.92E-25), and for genes expressed by granulocytes (P < 2.68E-56). We further validated expression and regulation of prioritized lncRNA upon IL-1β exposure in differentiated Caco-2 cells. Finally, we identified significant correlations between LINC01272 and CDKN2B-AS1 expression, and more severe mucosal injury.Conclusions:
We are the first to characterize a widespread dysregulation of 546 lncRNA in the ileum of treatment naïve pediatric CD patients. Unsupervised and supervised classifications using lncRNA showed comparable patients' grouping as the protein-coding genes, linking lncRNA to CD pathogenesis and their ability to predict disease state. Those lncRNA, after mechanistic exploration, may serve as potential new targets for RNA-based interventions for inflammatory and metabolic abnormalities in CD.