Research On Mechanism Of Insulin Production And Secretion Regulated By MiR-30d

microRNAs (miRNAs) are non-coding RNA with22nt in length that play a role indevelopment, cell differentiation and proliferation, apoptosis, cancer and other diseases atpost-transcription level. Reported miRNAs regulate diabetes process by targeting genesimportant for pancreas development, beta cell proliferation, insulin production, secretion,while the mechanisms by which miRNAs regulate insulin production and secretion are notclear. miRNAs modulate insulin signaling by gene regulation, so currently lots of biologicalstudies are fucosing on miRNA gene regulation to targets and related deseases.In this study, we employed MIN6and db/db mice to evaluate effects that miR-30dregulates target and signaling and mechanism of insulin production and secretion. From theexperiments, the following results were obtained:1. In situ Hybridization and real-time PCR were performed to measure miR-30d level indb/db mouse. miR-30d is down regulated in diabetic mice, while let-7which is related toapoptosis increases dramatically. Dual-luciferase experimental data showed thatoverexpression of miR-30d increased RIP-driven luciferase activity. These resultsdemonstrated that miR-30d plays a role in development diabetes by promoting insulin genetranscription.2. The ensuring effect of miR-30d upon the expression of MafA was examined bywestern blot and real-time PCR. It shows that the expression of MafA in high glucoseincreases and overexpression of miR-30d increased MafA transcription and expression.Immunofluorescent experiment shows miR-30d overexpression activates MafA proteinexpression at the single cell level. Using real-time PCR analysis the effect of miR-30doverexpression on MafA, the results show that overexpression of miR-30d promotestranscription of MafA and insulin. Western blot analysis shows miR-30d increases MafAexpression.3. Effects of miR-30d and TNF-α on MafA、Pdx-1and IRS2were detected by westernblot. It shows that TNF-α significantly inhibited MafA, Pdx-1and IRS2expression, whileoverexpression of miR-30d decreased in partial inhibition of MafA and IRS2. Inhibition of miR-30d induced further suppression of MafA and IRS2by TNF-α. Insulin secretion wasmeasured in MIN6cells with the same treatment. We observed that insulin secretion wassignificantly impaired by TNF-α, and that overexpression of miR-30d could only partiallyrestore this impairment.4. The effect of miR-30d on the expression of Map4k4was tested by western blot.Overexpression or miR-30d decreased the transcription and expression ofMap4k4.Overexpression of miR30d decreased the Map4k4-WT reporter activity, while theluciferase activity of point mutations in Map4k4-miR30d binding site (Map4k4-mut)doesn’t decrease. It shows that Map4k4is a direct target of miR-30d.In situ hybridizationanalysis with pancreas sections of diabetic mice was performed to measure Map4k4proteinand miR-30d level. Map4k4expression was significantly increased in the diabetic islets,while miR-30d decreased dramaticlly.The isolated islets were further analyzed for theexpression of Map4k4by Western blot and real-time PCR, and the results demonstrated thatboth Map4k4protein and mRNA levels were increased.5. We confirmed that both Map4k4protein and mRNA level were increased in MIN6cells treated by TNF-α. It shows TNF-α induces transcription and expression of Map4k4.The effects of silencing Map4k4on insulin, MafA, Pdx-1and IRS2was tested and datashow that silencing of Map4k4was able to restore pre-insulin2, MafA and IRS2expressionsignificantly, but no effect on Pdx-1.This study illustrated that miR-30d up-regulates insulin transcription factor via targetingMap4k4and furtherly promotes insulin gene expression and secretion. miR-30d can decreaseTNF-α-induced suppression of insulin transcription, production and secretion. The dataillustrate that the multi-functional miR-30d reveals the mechanism of diabetes developmentand provides a potential novel therapeutic target in the interventional approach to treatment ofdiabetes.