P089 Long ncRNA Landscape in the Rectum of Treatment Naïve Early Onset Ulcerative Colitis Highlights Association with Severity and Early and Late Disease Outcome, with Potential Role in Epithelial Metabolic Functions
Haberman Ziv, Y.(1);Braun, T.(1);Sosnovski , K.(1);Amir , A.(1);VanDussen , K.(2);Igor Ulitsky , I.U.(3);Griffiths , A.(4);Walters , T.(4);Mack , D.(5);Boyle, B.(6);Kugathasan, S.(7);Jegga, A.(8);Hyams , J.(9);Denson, L.(2);
(1)Tel-HaShomer Sheba Medical Center, Department of Pediatric Gastroenterology, Ramat Gan, Israel;(2)Cincinnati Children's Hospital Medical Center, Gastroenterology, Cincinnati, United States;(3)Weizmann Institute of Science, Biology, Rehovot, Israel;(4)Hospital For Sick Children, Pediatrics, Toronto, Canada;(5)Children's Hospital of East Ontario, Pediatrics, Ottawa, Canada;(6)Nationwide Children's Hospital, Pediatrics, Colombus, United States;(7)Emory University, Pediatirics, Atlanata, United States;(8)Cincinnati Children's Hospital Medical Center, Pediatirics, Cincinnati, United States;(9)Connecticut Children’s Medical Center, Pediatrics, Hartford, United States; PROTECT cohort
Background
Long non-coding RNAs (lncRNAs) are tissue-specific and regulate cellular functions. We previously reported a dramatic inhibition of epithelial metabolic mitochondrial functions in mucosal biopsies from the UC PROTECT cohort. We aimed to define lncRNAs that are dysregulated in UC, linked with attenuated epithelial metabolic functions, and associated with worse outcome.
Methods
Transcriptomics of pre-treatment rectal biopsies in PROTECT UC [206 UC and 20 controls (Ctl)]. Weighted gene co-expression network analysis (WGCNA) to identify modules and gens associated with UC severity, week 4 remission after 5-ASA/steroids (W4R), week 52 steroid-free remission (W52SFR), and colectomy within 3 years. Epithelial mechanistic data of prioritized lncRNA.
Results
PROTECT transriptomics included 2,826 lncRNA genes with TPM>1 in 20% of samples. Principal components analysis (PCA, Fig 1) using these lncRNA showed distinct clusters of UC and Ctl, and PC2 values were linked (p<0.01) with endoscopic (Mayo score) and clinical (PUCAI) disease severities. Random forest classifiers using the 2,826 lncRNA trained to distinguish UC from Ctl using PROTECT, predicted the correct diagnosis in PROTECT (AUC=1) and in RISK (AUC=0.88) connecting lncRNA to UC pathogenesis in two independent cohorts. WGCNA on the 2,826 lncRNA resulted in 6 modules (M1-M6) associated with UC (p<0.01, Fig 2). M1, which contained lncRNA reduced in UC, was associated with lessW4R (p=0.01) less W52SFR (p=0.04) and increased colectomy (p=0.02). In contrast, M6, which contained lncRNA elevated in UC, was linked with W4R (p=0.008). The hub genes in the M1 and M6 modules included GATA6-AS1 and LINC01272 lncRNAs, respectively. Re-analysis of single cell datasets supported epithelial expression of GATA6-AS1 and myeloid expression of LINC01272. GATA6-AS1 co-expression with protein-coding genes in PROTECT showed significant enrichment for mitochondrial functions (p<1E-40). Reduction of GATA6-AS1 expression with 2 shRNAs resulted in reduced mitochondrial complex IV MT-CO2 mRNA and protein and reduction of LGR5, linking mitochondrial genes and function with epithelial renewal. We recently showed pronounced reduction of the mitochondrial membrane potential (MMP) by JC-1 staining in UC epithelia. Consistent with this finding, GATA6-AS1 inhibition reduced MMP levels from 0.22 in control to 0.08 (p=0.01), similarly to MMP of cells treated with CCCP that is known to depolarize and reduce MMP.
Conclusion
LncRNA are significantly linked with UC and disease severity, and the M1 epithelial enriched module shows significant association with worse outcome. GATA6-AS1 within the M1 hub genes show potential role in epithelial metabolic functions and renewal.