Abstract
MicroRNAs (miRs) are short RNA nucleotide sequences that regulate protein synthesis by cleaving messenger RNA (mRNA) or preventing their translation. MiRs are synthesized by a highly specialized process involving protein complexes such as DROSHA and Dicer, RNA polymerases and Argonaut proteins (RISC complex). Some miRs are associated with PDX1, Neurog3 and mafA proteins, transcription factors essential for proper pancreatic beta cell development and function. These miRs may inhibit, stimulate or even be stimulated by these transcription factors. PDX1 is responsible for differentiating, in the embryonic period, progenitor pancreatic cells in endocrine and exocrine lines; after that, PDX1 is down-regulated and is only up-regulated again at birth and is related to the general functioning of the pancreatic beta cell. The differentiation and specification of endocrine lineage derives from the expression of Neurog3, an event that leads to the emergence of the beta cell in the embryonic period; however, after performing this role, such protein is almost completely inhibited by miRs 15-a, 15-b, 16 and 195. Beta-cell maturation and insulin gene expression is due to mafA expression in the end of pregnancy and gradually increases after birth due to miR-30d stimulation. The KSRP protein is part of the structure of the DROSHA and Dicer protein complexes. If this transcription factor suffers SUMOylation, its exportation is stimulated and the synthesis of miRs decreases. In addition, KRSP appears to be associated with insulin secretion and glycemic metabolism. The purpose of this research is to perform KSRP knocked-down in INS-1E pancreatic beta cells, with the following objectives: 1) to observe if there is alteration of miRs expression associated with PDX1, Neurog3 and mafA transcription factors by PCR evaluation TaqMan fluorescent probe system; 2) determine if there is a decrease or increase in transcription factors performed by Western Blotting; and 3) whether there is a change in insulin transcription and biosynthesis processes by quantitative PCR and Western Blotting. This project may contribute to the elucidation of mechanisms essential for the synthesis and regulation of beta cell transcription factors and, therefore, the development of possible therapeutic strategies against beta cell dysfunction and type 2 diabetes mellitus.
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