GATA6-AS1 Regulates Intestinal Epithelial Mitochondrial Functions, and its Reduced Expression is Linked to Intestinal Inflammation and Less Favourable Disease Course in Ulcerative Colitis

BACKGROUND AND AIMS: Widespread dysregulation of long non-coding RNAs [lncRNAs] including a reduction in GATA6-AS1 was noted in inflammatory bowel disease [IBD]. We previously reported a prominent inhibition of epithelial mitochondrial functions in ulcerative colitis [UC]. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not defined. METHODS: Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment-naïve independent human cohorts [n=673]. RNA pull-down followed by mass spectrometry was used to determine the GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2 cells were used to elaborate on GATA6-AS1 functions. RESULTS: GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn\u27s disease [CD] ileum and UC rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to a more severe UC form, and to a less favourable UC course. The GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in coeliac disease that is induced in UC, CD and coeliac disease, in contrast to GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with a reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in a reduction of metabolites linked to aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1-deficient cells rescued mitochondrial respiration. CONCLUSIONS: GATA6-AS1 levels are reduced in UC, CD and coeliac disease, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels

Reduced LHFPL3-AS2 lncRNA expression is linked to altered epithelial polarity and proliferation, and to ileal ulceration in Crohn disease

Abstract Disruption of intestinal epithelial functions is linked to Crohn disease (CD) pathogenesis. We identified a widespread reduction in the expression of long non-coding RNAs (lncRNAs) including LHFPL3-AS2 in the treatment-naïve CD ileum of the RISK pediatric cohort. We validated the reduction of LHFPL3-AS2 in adult CD and noted a further reduction in patients with more severe CD from the RISK cohort. LHFPL3-AS2 knockdown in Caco-2 cells robustly affected epithelial monolayer morphogenesis with markedly reduced confluency and spreading, showing atypical rounding, and clumping. mRNA-seq analysis of LHFPL3-AS2 knockdown cells highlighted the reduction of genes and pathways linked with apical polarity, actin bundles, morphogenesis, and the b-catenin-TCF4 complex. LHFPL3-AS2 knockdown significantly reduced the ability of cells to form an internal lumen within the 3-dimensional (3D) cyst model, with mislocalization of actin and adherent and tight junction proteins, affecting epithelial polarity. LHFPL3-AS2 knockdown also resulted in defective mitotic spindle formation and consequent reduction in epithelial proliferation. Altogether, we show that LHFPL3-AS2 reduction affects epithelial morphogenesis, polarity, mitotic spindle formation, and proliferation, which are key processes in maintaining epithelial homeostasis in CD. Reduced expression of LHFPL3-AS2 in CD patients and its further reduction with ileal ulceration outcome, emphasizes its significance in this context

GATA6-AS1 regulates intestinal epithelial mitochondrial functions, and its reduced expression is linked to intestinal inflammation and less favorable disease course in ulcerative colitis (UC)

Background and aims: Widespread dysregulation of long non-coding RNAs (lncRNAs) including a reduction in GATA6-AS1 was noted in inflammatory bowel disease (IBD). We previously reported a prominent inhibition of epithelial mitochondrial functions in UC. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not yet defined. Methods: Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment naïve independent human cohorts (n=673). RNA pull-down followed by mass-spectrometry was used to determine GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2, were used to elaborate on GATA6-AS1 functions. Results: GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn disease (CD) ileum and in ulcerative colitis (UC) rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to more severe UC form, and to less favorable UC course. GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in celiac disease that is induced in UC, CD, and celiac, in contrast GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in reduction of metabolites linked with aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1 deficient cells rescued mitochondrial respiration. Conclusions: GATA6-AS1 levels are reduced in UC, CD, and celiac, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 level

OrganoID: A versatile deep learning platform for tracking and analysis of single-organoid dynamics

Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses. However, these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (95%) and size (97%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified strong dose effect sizes on organoid circularity, solidity, and eccentricity. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications