P055 Increased Matrix stiffness Modulates MicroRNA expression and fibrogenesis in an in vitro model of intestinal fibrosis
L.A. Johnson1, E. Rodansky1, 2, P.D. Higgins*1
1University of Michigan, Internal Medicine - Gastroenterology, Ann Arbor, MI, United States, 2University of Michigan, Internal Medicine - Gastroenterology, Ann Arbor, United States
Fibrosis is the common endpoint of organ failure in many organ systems and disease states, including the intestine and Crohn's disease (CD). In colonic myofibroblasts, extracellular matrix stiffness characteristic of CD can activate fibrogenesis. microRNAs, small non-coding RNAs (miRNAs), have been shown to regulate fibrosis by targeting and fine-tuning gene expression. We postulated that pathological matrix stiffness may regulate fibrogenesis in vitro by modulation of miRNAs.
Normal human colonic myofibroblasts (Ccd-18co cells) were cultured on either physiologically soft or pathologically stiff matrices for 48 hours. Candidate stiffness responsive miRNAs were identified by screening a fibrosis-specific miRNA array. Stiffness responsiveness was confirmed by QPCR using individual miRNAs. miRNAs mimics were used to determine whether miRNAs alone induce fibrogenic gene expression.
Eight stiffness-responsive miRNAs were identified and subsequently validated by QPCR. Of interest, two bicistronic miRNAs, miR-143 and miR-145, which are two key regulators of intestinal smooth muscle were strongly induced (9.5 and 7-fold respectively) by high matrix stiffness. Transfection of Ccd-18co cells grown on low stiffness (4.3 kPa) with miR-143 and miR-145 mimics recapitulated the fibrogenic effects of high matrix stiffness. Stiffness-responsive fibrogenic genes fibronectin (FN1), myosin light chain kinase (MYLK) and smooth muscle actin (ACTA2) were induced while the pro-inflammatory gene COX-2 (PTGS2) was repressed.
Pathological matrix stiffness induces pro-fibrogenic miRNAs in normal human myofibroblasts. Addition of miRNAs mimics to cells in a normal physiologically soft (compliant) environment, recapitulates the effects of increased matrix stiffness on fibrogenic gene expression. miRNAs may be a novel mechanism for regulation/auto-propagation of matrix stiffness-mediated intestinal fibrosis in Crohn's disease.