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Inhibition of miR-7 in developing pancreas and in cultured pancreatic buds affects beta cell differentiation

Margarita Nieto, Pedro Hevia, Enrique Garcia, Dagmar Klein, Silvia Alvarez-Cubela, Valia Bravo-Egana, Samuel Rosero, R.Damaris Molano, Nancy Vargas, Camillo Ricordi, Antonello Pileggi, Juan Diez, Juan Domínguez-Bendala, Ricardo Pastori

Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, FL, United States

miRNAs are small non-coding gene products that negatively regulate gene expression post-transcriptionally. MiRNAs control key biological events such as embryonic development and organ differentiation, including pancreatic specification and islet function. miR-7 is highly expressed in human fetal and adult pancreatic endocrine cells. We studied the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic day e10.5 and e11.5, then began to increase at e13.5 through e14.5 and eventually decreased by e18. Endocrine marker Isl1 expression co localizes with miR-7 suggesting that miR-7 is expressed preferentially in endocrine cells. Whole mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube as well. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall down-regulation of insulin production, decreased beta cell numbers and glucose intolerance in the post natal period. The cell death assessment by TUNEL assay of in vivo treated samples showed extensive cell death throughout the pancreatic tissue in MO anti-miR-7 injected animals but not in the MO controls. Because of the high miR-7 expression in the embryonic brain and the fact that all cell types were affected equally, we assume that a systemic effect is the major contributor.

On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds led to beta cell death and generation of beta cells expressing less insulin than those in MO control. Therefore, besides the potential indirect effect on pancreatic differentiation derived from miR-7 systemic downregulation, its knockdown in vitro proves to have a beta cell-specific effect as well.