miR-296-3p, miR-298-5p and their downstream networks are causally involved in the higher resistance of mammalian pancreatic α cells to cytokine-induced apoptosis as compared to β cells

Background: The molecular bases of mammalian pancreatic alpha cells higher resistance than beta to proinflammatory cytokines are very poorly defined. MicroRNAs are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus. Results: Through high-throughput real-time PCR, we analyzed the steady state microRNA transcriptome of murine pancreatic alpha (alpha TC1-6) and beta (beta TC1) cells: their comparison demonstrated significant differences. We also characterized the alterations of alpha TC1-6 cells microRNA transcriptome after treatment with proinflammatory cytokines. We focused our study on two microRNAs, miR-296-3p and miR-298-5p, which were: (1) specifically expressed at steady state in alpha TC1-6, but not in beta TC1 or INS-1 cells; (2) significantly downregulated in alpha TC1-6 cells after treatment with cytokines in comparison to untreated controls. These microRNAs share more targets than expected by chance and were co-expressed in alpha TC1-6 during a 6-48 h time course treatment with cytokines. The genes encoding them are physically clustered in the murine and human genome. By exploiting specific microRNA mimics, we demonstrated that experimental upregulation of miR-296-3p and miR-298-5p raised the propensity to apoptosis of transfected and cytokine-treated alpha TC1-6 cells with respect to alpha TC1-6 cells, treated with cytokines after transfection with scramble molecules. Both microRNAs control the expression of IGF1R beta, its downstream targets phospho-IRS-1 and phospho-ERK, and TNF alpha. Our computational analysis suggests that MAFB (a transcription factor exclusively expressed in pancreatic alpha cells within adult rodent islets of Langerhans) controls the expression of miR-296-3p and miR-298-5p. Conclusions: Altogether, high-throughput microRNA profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of miR-296-3p and miR-298-5p, coupled to upregulation of their targets as IGF1R beta and TNF alpha, is a major determinant of mammalian pancreatic alpha cells resistance to apoptosis induction by cytokines.