e., pHi alkalinization upon Cl removal) (which forces AE2 to this website operate in a reverse mode, extruding intracellular Cl− in exchange with HCO) and further normalization of pHi after restoration of extracellular Cl− (which allows AE2 to operate in a physiological mode secreting HCO). The rates of initial alkalinization and subsequent pHi recovery obtained by these maneuvers were both markedly decreased in H69 cholangiocytes transfected with pre-miR-506 versus control transfected cholangiocytes (Fig. 4A,B). These data indicate that overexpression of miR-506 leads to decreased AE2 activity in human cholangiocytes. Also, we investigated the effect of miR-506
on the hydrocholeretic function of human cholangiocytes using the model of 3D-cultured H69 cholangiocytes. Under 3D conditions, H69 human cholangiocytes may form cystic structures, which spontaneously expand over time as a consequence of fluid secretion into the cyst lumen. As previously reported for 3D-cystic p38 MAPK cancer structures derived
from rat cholangiocytes,32 the expansion rate of the human H69 cystic structures was accelerated by the presence of secretin in culture medium during 30 minutes (6.40% ± 1.26%; P = 0.0002; Fig. 5), indicating an increase in fluid secretion to the cyst lumen. But, preincubation with pre-miRNA-506 in culture medium for 24 hours blocked the secretin-stimulated expansion of H69 cholangiocyte cystic structures. Altogether,
our findings indicate that up-regulated miR-506 may inhibit both AE2 expression and AE2-mediated hydrocholeretic function in human cholangiocytes. Next, we investigated whether our previous data of decreased AE2 activity in cultured PBC cholangiocytes16 could be related to overexpression of miR-506. We isolated PBC cholangiocytes using pieces of 上海皓元医药股份有限公司 a liver explant from a female patient with PBC and cultured them for 7-10 passages. Also, we isolated normal cholangiocytes from liver pieces of normal bordering tissue (obtained during a surgical intervention in a female patient; Supporting Materials) and cultured them as for PBC cholangiocytes. qPCR analysis of miR-506 levels revealed a 2-fold increase in cultured PBC cholangiocytes versus normal cholangiocytes (Fig. 6A). However, expression levels of two other miRNAs predicted to potentially target human AE2 mRNA (i.e., miR-149-3p and miR-765) were down-regulated in cultured PBC cholangiocytes, compared to normal cholangiocytes (Supporting Materials and Supporting Fig. 2). Microfluorimetric studies indicated that miR-506 overexpression in PBC cholangiocytes was associated with decreased AE2 activity (Fig. 6B). To test the hypothesis that the down-regulated AE2 activity in PBC cholangiocytes is at least partially the result of the increased miR-506 levels, we used commercial anti-miR-506 oligonucleotides to inhibit miR-506.