| ObjectivePancreatic β-cell dysfunction is a central component of the pathogenesis of diabetes. miRNAs have become one of the most encouraging and fruitful fields in biological research and have been implicated as new players in the pathogenesis of diabetes and diabetes-associated complications. The role of miRNAs in diabetes starts as early as the development of pancreatic islets. Recently, many miRNAs have been reported to play essential roles in many aspects of diabetes. FGF-21enhances glucose uptake in adipocytes, protects from diet-induced obesity when overexpressed in transgenic animals, and lowers blood glucose and triglyceride levels when administrated to diabetic animals and is a good way to treat diabetes. But the mechanism of regulation of FGF-21is not known. FGF-21is predicted as a direct gene of miR-577by bioinforamtic analysis.Then if miR-577effects insulin secretion in β-cell by regulating FGF-21?And the function and mechanism of miR-577in pancreatic β-cell are unknown.there are few studies in these field in the world now.So we are going to identify the relation between miR-577and FGF-21,and to study how does miR-577regulate FGF-21in Pancreatic β-cell,and then further to explore the function and mechanism of miR-577in Pancreatic p-cell.Methods1. Predict that FGF-21is a direct gene of miR-577by bioinforamtic analysis. To prove our hypothesis and check whether FGF-21is a direct gene of miR-577.we use two ways to confirm our results by overexpressed miR-577or inhibition of miR-577.(1)Construct wild type and mutant of luciferase reporter plasmids of3’-UTR of the FGF-21gene,then co-transfect293T-cell specialty with either oligonucleotides of miR-577or of miRNA-control (miR-577group and miR-control group).And then we use luciferase assay to analyses luciferase activity48h later.(2)Construct LV-Puro-miR-577recombinded Vector and empty LV-Puro Vector,then infect either embryonic pancreatic β-cell(EPB) or INS-1β-cell of mice(INS-1)(miR-577group and miR-control group),we use real-time RT-PCR to analyse expression of FGF-21gene mRNA48h later.(3)The same method to infect either EPB cell or INS-1cell(miR-577group and miR-control group),then use ELISA to analyse content of FGF-2148h later.2. Use insulin secretion assay to check whether miR-577is involved in insulin secretion by target FGF-21.(1) EPB cells and EPB cells infected with LV-miR-577or LV-miR-control were treated for three different time points (0,24, and48h) with50nmol/1FGF-21before exposure to15mmol/l glucose for90min(FGF-21group, miR-577+FGF-21group,and miR-control+FGF-21group),then use ELISA to analyse insulin content.(2)EPB cells and EPB cells infected with LV-miR-577or LV-miR-control were treated for two different time points (0,24h) with50nmol/l FGF-21before exposure to15mmol/1glucose for90min(miR-577+FGF-21group,miR-control+FGF-21group and FGF-21group),then use real-time RT-PCR to analyse expression of insulin gene mRNA.(3)EPB cells and EPB cells infected with LV-miR-577or LV-miR-control were treated for seven different time points (0,4,8,12,24,48and72h) with50nmol/1FGF-21before exposure to15mmol/l glucose for90min,then use ELISA to analyse insulin content to observe the change of insulin content.3. We check whether miR-577regulates FGF-21activated signaling pathway by western-blotting assay and ELISA in EBP cells.(1)EPB cells and EPB cells infected with LV-miR-577or LV-miR-control were treated for three different time points (40,80and72min) with50nmol/1FGF-21.then use ELISA to analyse ERK phosphorylation.(2)EPB cells and EPB cells infected with LV-miR-577or LV-miR-control were treated for three different time points (40,80and72min) with50nmol/l FGF-21,then use western-blotting assay to analyse ERK phosphorylation.Results1. FGF-21is a direct target gene of miR-577(1) In cells transfected by wild type of luciferase reporter plasmids of3’-UTR of the FGF-21gene, The luciferase activity of miR-577group was much lower than miR-control group(0.20±0.226vs0.60±0.234)(P<0.01); wile in cells transfected by mutant type of luciferase reporter plasmids of3’-UTR of the FGF-21gene, there was not difference between the two group(1.10±0.512vs1.00±0.326)(P>0.05).(2) The expression of FGF-21gene mRNA of miR-577group was much lower than that of miR-control group in both EPB cells and INS-1cells(0.38±0.345vs1.00±0.231;0.41±0.357vs1.02±0.523)(both P<0.01).(3) The content of FGF-21of miR-577group was much lower than that of miR-control group in both EPB cells and INS-1cells(350±10.87vs1210±50.01,420±20.95vs1550±61.56)(both P<0.01).2. MiR-577inhibit insulin secretion in EPB cells by negatively regulating FGF-21(1) Before incubation with FGF-21(0h),insulin content of miR-577+FGF-21group was lower than that of FGF-21group and also lower than that of miR-control+FGF-21group(0.286±0.026vs0.420±0.050,0.286±0.026vs0.410±0.049),but there was not statistical significance(both P>0.05);while there was not difference between miR-control+FGF-21group and FGF-21group(0.410±0.049vs0.420±0.050)(P>0.05).(2) At time points of24h and48h after incubation with FGF-21, we use glucose to stimulate for90minutes, insulin content of miR-577+FGF-21group was much lower than that of FGF-21group(3.143±0.517vs6.850±0.523,3.132±0.265vs6.250±0.775)(both P<0.01); and also much lower than that of miR-control+FGF-21group(3.143±0.517vs6.340±0.045,3.132±0.265vs6.139±0.052)(both P<0.01); while there was not difference between FGF-21group and miR-control+FGF-21group(6.850±0.523vs6.340±0.045,6.250±0.775vs6.139±0.052)(both P>0.05).(3) Before incubation with FGF-21(0h),the expression of FGF-21gene mRNA of miR-577+FGF-21group was lower than that of FGF-21group and also lower than that of miR-control+FGF-21group(0.305±0.056vs0.616±0.035,0.305±0.056vs0.609±0.227), but there was not statistical significance(both P>0.05);while there was not difference between miR-control+FGF-21group and FGF-21group(0.609±0.227vs 0.616±0.035)(P>0.05).(4) At time point of24h after incubation with FGF-21,we use glucose to stimulate for90minutes, the expression of FGF-21gene mRNA of miR-577+FGF-21group was lower than that of FGF-21group(0.795±0.156vs1.750±0.186)(P<0.01); and also much lower than that of miR-control+FGF-21group.(0.795±0.156vs1.715±0.164)(P<0.01); While there was not difference between FGF-21group and miR-control+FGF-21group(1.750±0.186vs1.715±0.164)(P>0.05).(5)EPB cells infected with LV-miR-577elevated insulin content levels,After8h incubation with FGF-21, the increase of insulin content reached its maximum (230%) and remained relatively constant for up to72h of exposure to FGF-21. But the insulin content of the EPB cells infected with LV-miR-577were lower than the EPB cells infected with LV-miRNA-control at time points of0h,4h,8h,12h,24h,48h and72h after incubation with50nmol/1FGF-21and stimulation with15mmol/1glucose for90minutes.3.MiR-577inhibits FGF-21activated FGF receptor downstream signaling cascades in β-cells(1) At time points of40,80and120minutes,ERK1/2phosphorylation activity of EPB cells infected with LV-miR-577in miR-577+FGF-21group was weaker than that of EPB cells in FGF-21group and miR-control group(0.14±0.123vs2.15±0.145,0.98±0.125vs1.62±0.214,0.83±1.785vs1.41±0.189)(all P<0.05);(0.14±0.123vs2.14±0.256,0.98±0.125vs1.61±0.326,0.83±1.785vs1.42±0.212)(all P<0.05);While there were not differences between miR-control group and FGF-21group(2.14±0.256vs2.15±0.145,1.61±0.326vs1.62±0.214,1.42±0.212vs1.41±0.189)(all P>0.05).(2) ERK1/2phosphorylation activity of cells both in FGF-21group and miR-control group got enhanced, and reached their maximum at40minute after stimulated of50nmol/1FGF-21, then weakened with time prolonged.But ERK1/2phosphorylation activity of EPB cells infected with LV-miR-577in miR-577+FGF-21group was weaker than that of EPB cells in FGF-21group and than miR-control group as measured by Western blot.Conclusion 1. MiR-577is a direct target gene of FGF-212. MiR-577inhibits insulin secretion in EPB cells by negatively regulating FGF-213. MiR-577inhibits FGF-21activated FGF receptor downstream signaling cascades in β-cells.In summary, our data showed that miR-557is a novel regulator of FGF-21, and inhibits insulin secretion and inactive FGF-21downstream signal pathway. |
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