Confluence does not affect the expression of miR-375 and its direct targets in rat and human insulin-secreting cell...

MicroRNAs are small non-coding RNAs, which negatively regulate the expression of target genes. They have emerged as important modulators in beta cell compensation upon increased metabolic demand, failure of which leads to reduced insulin secretion and type 2 diabetes. To elucidate the function of miRNAs in beta cells, insulin-secreting cell lines, such as the rat insulinoma INS-1 832/13 and the human EndoC-βH1, are widely used. Previous studies in the cancer field have suggested that miRNA expression is influenced by confluency of adherent cells. We therefore aimed to investigate whether one of the most enriched miRNAs in the pancreatic endocrine cells, miR-375, and two of its validated targets in mouse, Cav1 and Aifm1, were differentially-expressed in cell cultures with different confluences. Additionally, we measured the expression of other miRNAs, such as miR-152, miR-130a, miR-132, miR-212 and miR-200a, with known roles in beta cell function. We did not see any significant expression changes of miR-375 nor any of the two targets, in both the rat and human beta cell lines at different confluences. Interestingly, among the other miRNAs measured, the expression of miR-132 and miR-212 positively correlated with confluence, but only in the INS-1 832/13 cells. Our results show that the expression of miR-375 and other miRNAs with known roles in beta cell function is independent of, or at least minimally influenced by the density of proliferating adherent cells, especially within the confluence range optimal for functional assays to elucidate miRNA-dependent regulatory mechanisms in the beta cell.