LONG NONCODING RNA GAS5 REGULATES INTESTINAL EPITHELIAL BARRIER FUNCTION BY ALTERING ACTIVITY OF RNA-BINDING PROTEIN HUR

Gut barrier dysfunction occurs commonly in various pathologies, but the exact mechanism underlying the barrier disruption remains largely unknown. Mammalian genomes transcribe a vast number of noncoding RNAs (ncRNAs) with active roles in gene regulation. Long ncRNAs (lncRNAs) are defined as transcripts spanning >200 nucleotides in length and function as molecular scaffolds, decoys, or signals to control virtually every level of gene regulation. LncRNA GAS5 participates in different cellular processes and is involved in diverse human diseases. In this study, we examined the role of lncRNA GAS5 in modulating the intestinal epithelial barrier function. Methods: Intestinal mucosal tissues were collected from mice treated with 3% dextran sodium sulfate (DSS) in drinking water for 5 days or exposed to cecal ligation and puncture (CLP) for 24 h and patients with inflammatory bowel diseases (IBD). Caco-2 cells were used for an in vitro intestinal epithelial permeability model. Primary enterocytes were isolated from the small intestine of mice for deriving intestinal organoids. Levels of cellular GAS5 were elevated by transfection with a vector that expressed GAS5 but decreased by transfection with specific small interfering RNA targeting GAS5 (siGAS5). Gut barrier function was detected by tracer FITC-dextran and transepithelial electrical resistance (TEER) assays. Levels of lncRNAs and proteins were examined by qPCR, immunoblotting, and immunofluorescence assays. Results: Compared with controls, mucosal GAS5 abundances increased significantly in the colons of DSS-treated mice and in the small intestine of CLP-mice. Increased levels of GAS5 in the mucosa of CLP-mice were associated with an increase in gut permeability. Similarly, mucosal levels of GAS5 also increased remarkably in human intestinal tissues from IBD patients. Ectopically expressed GAS5 disrupted function of the intestinal epithelial barrier, as indicated by increased paracellular permeability of FITC-dextran and lowered TEER in an in vitro system. In contrast, decreasing the levels of cellular GAS5 by transfection with siGAS5 improved the epithelial barrier function. GAS5 overexpression also decreased the levels of tight junction (TJ) proteins, including claudin-1, -2, -3, and ZO-1, in both Caco-2 cells and intestinal organoids, but it failed to alter JAM-1, occludin, and E-cadherin content. Mechanistically, induced GAS5 decreased levels of RNA-binding protein HuR, thus preventing an HuR-mediated increase in the stability and translation of mRNAs encoding various TJ proteins. Conclusions: These results indicate that 1) GAS5 downregulates intestinal epithelial barrier function and 2) GAS5 inhibits expression of TJs at least partially by inactivating HuR function. These findings highlight a novel role of increased GAS5 in the pathogenesis of gut barrier dysfunction.