FIBROBLAST ACTIVATION PROTEIN IS OVEREXPRESSED IN INFLAMED INTESTINAL MUCOSA AND MAY BE MODULATED BY MIR-21

ANAHI SORIA RENDON; EMANUEL BARBIERA; FERREYRA COMPAGNUCCI MALENA; MARIA BELEN POLO; MANUELA ILID; ANDRES ROCCA; ALICIA SAMBUELLI; JUAN ARRIOLA; MARIA BELEN SARACHO; KARINA COLLIA; MARIA VIRGINIA GENTILLI; GABRIEL GONDOLESSI; GUSTAVO J. CORREA; MARTIN YANTORO; MARIA VICTORIA MERCADER; JULIO DE MARIA; MARTIN RUMBO; GUILLERMO H. DOCENA; CECILIA ISABEL MUGLIA; RENATA CURCIARELLO

San Luis

Congreso; Reunión Anual de Sociedades de Biociencia SAI 2023; 2023

Fibroblast activation protein (FAP) is a transmembrane endopeptidase present in cancer associated fibroblasts, which contributes to extracellular matrix remodeling and mesenchymal cell activation, favoring tumoral growth. FAP overexpression has been reported in lung and breast cancer, under microRNA-21 (miR-21) regulation. Nevertheless, FAP role is not completely described in colorectal cancer (CRC). We previously showed miR-21 overexpression in inflammatory bowel disease (IBD) patients´ mucosa and intestinal fibroblasts. IBD is a predisposing condition for CRC development; we aim to investigate FAP expression in the colonic mucosa of patients with chronic intestinal inflammation.Colonic biopsies and surgical samples were taken from macroscopically inflamed and uninflamed mucosa from patients with IBD, CRC, polyps, or healthy controls (HC). Mucosal pieces were fixed and staining for FAP and α-SMA was performed by indirect immunofluorescence (IF) .Total RNA was extracted from mucosal samples, and retro-transcribed. qPCR was performed on cDNA using specific primers for miR-21, fap, α-sma and HPRT.Relative expression was normalized to U6 for miRNA or to HPRT for the rest, and data were analyzed using the comparative threshold method (2−ΔCt). Fibroblast primary cultures were established after isolating lamina propria cells by mechanical and enzymatic digestion of biopsies or tissue sections. Cells were cultured in DMEM Glutamax 20% FBS with antibiotics. Fibroblast culture supernatants were collected and ultracentrifuged at 100.000xg for 2 h for exosome enrichment, which were visualized by atomic force microscopy. Mi-R21 expression was evaluated by qPCR in cells and exosome fractions. In vitro induction of FAP was evaluated on fibroblasts by IF, after stimulation with TGF-β (1 and 10 ng/ml) or exosomal fraccion for 24 h. Images were acquired with Leica SP5 confocal microscope. FAP and α-SMA merge images were analyzed with Image J and QuPath softwares.We observed an overexpression of miR-21 and its target genes in the intestinal mucosa and fibroblasts from IBD and CRC patients, compared to HC (p