Epigenetics Regulate The Development And Function Of Pancreaticβ Cells As Well As Diabetes Onset

Background:Diabetes is a group of chronic metabolic diseases in which a person has high blood glucose level because the pancreatic islets can not produce enough insulin, cells do not respond to the insulin normally or both. High blood glucose levels can cause several classical symptoms, including:polyuria, polydipsia and polyphagia. Chronic diabetes can significantly enhance the risk of long-term complications such as atherosclerosis. Diabetic retinopathy. chronic kidney disease and diabetic foot ulcers. It is believed genes and environmental factors interact to cause diabetes, but the precise etiology of most cases of diabetes is still unknown. There are two main types of diabetes, type1and type2. Type1diabetes is characterized by loss of the insulin-producing β cells in the in the pancreatic islets, leading to insulin deficiency. The bata cell loss is cuased by either autoimmune destruction or idiopathic susceptibility. Many different genes contribute to its onset, so finding the mechanisms that regulate the diabetes-relavent gene expression is of grave importance to the cure of diabetes.Epigenetic changes can modify the expression of certain genes without changing sequence of DNA. There are several types of epigenetic inheritance systems methylation of DNA, RNA interference, histone acetylation and protein modification. MiRNAs are a class of21-25nucleotide single stranded noncoding small RNAs that function as important regulators of gene expression through inhibiting effective mRNA translation via imperfect base pairing with the3’-untranslated region (3’UTR) of target mRNAs in animals. MiRNAs are tissue-specific and considered to play important roles in cell function. Some microRNAs have shown their specific roles in β cell development and fuction. Histones are small proteins that, by complexing wtih DNA, form the nucleosome core. Repetitive units of this nucleosome led to the chromatin in which all the genome is packaged. Histone deacetylases (HDACs) make histones to wrap the DNA more tightly to inhibit gene transcription, thus to play a regulatory role in many physiological activities including insulin signaling.HDAC inhibitors have shown the effect of protecting β cells from cytokines in the inflammatory process. The effect of HDAC inhibitors and HDACs on β-cells remains unclear. We studied the direct effect of one HDACs inhibitor (TSA) on β cell lines and changed miRNA profiles induced by TSA, generated several miRNA knockout mice strains to observe the sensitivity to Streptozotocin (STZ) induced diabetes, generated βP cell specific HDAC3knockout mice to study the effect of HDAC3on β cell proliferation and function and hope to find the link between HDAC3and β cell as well as diabetes onset to provide theoretical basis for the research and development of drugs for the treatment of diabetes.Methods:1, Firstly MIN6cells were treated with different concentrations of TSA for different times, Cell viability was evaluated by the MTT-based cytotoxicity assay, Annexin V-APC and7-AAD staining for apoptosis analysis. miRNAs expression was performed using Taqman MicroRNA assay. Predict the changed microRNAs regulating β cell apoptosis pathway factors.2, Systemic miRNA155knockout mice and Tie2-Dicerfl/fl mice were generated to test the sensitivity to multiple low-dose streptozotocin-induced diabetes, thus illustrating the function of miRNAs in the onset of cytokine induced diabetes.3, To investigate the function of HDAC3in the development and function of β cells. Mice with HDAC3specifically depleted in β cells were generated, mice weight and blood glucose levels were monitored and function of islets were assessed using glucose tolerance test and glucose stimulated insulin secretion. β cell number and proliferation were measured by Histological sections and immunoflurescence, multiple low-dose STZ treatment was performed to test the sensitivity to cytokine induced β cell diabetes. HDAC3siRNA transfection was done in vitro to test the changes insulin relavent and HDAC3potential targeted gene expression using realtime PCR. Suppressor of cytokine signaling3(SOCS3) was one of the potential targets of HDAC3, we then used SOCS3and HDAC3double silence to test the insulin relative gene expression and insulin content and secretion.Results:1, Cell viability was inhibited as the increasing TSA concentration and the extending of treatment time,TSA significantly elevated cell apoptosis,61miRNAs were down-regulated and56miRNAs were up-regulated (|-ΔΔCt|≥1), among them12up-regulated and25down-regulated miRNAs target specific apoptosis pathway factors.2, Loss of miR155did not show any difference in the multiple low-dose streptozotocin induced diabetes incidence compared with wide type mice, but loss of total miRNAs in hematopoiesis prevents STZ-induced autoimmune diabetes.3, Mice with HDAC3depleted β cells showed marked decreased body weight and higher blood glucose level compared with littermate controls, some individuals even developed spontaneous diabetes. They exhibited glucose intolerance, impaired insulin secretion, there was no obvious decrease in the islets volume, but the β cell insulin content as well as proliferation dramatically decreased. They showed significantly enhanced sensitivity to STZ induced diabetes. HDAC3silencing in vitro significantly decreased the mRNA level of insulin, SOCS3was considered to be the potential target of HDAC3after screening all potential genes. CHIP confirmed the binding of HDAC3to the promoter of SOCS3, silencing SOCS3together with HDAC3can, at leat partially attenuated the inhibitive effect of HDAC3silencing to insulin production and secretion.Conclusions: 1, HDACs inhibitor TSA decreases β cell proliferation, increases apoptosis and altered miRNAs expression, and indicates the potential involvement of miRNAs in TSA mediated MIN6cell apoptosis. As a promising anti-cancer drug, these negative effects of TSA on β cells should also be considered.2, Unlike prevention in other autoimmune diseases, loss of miR-155does not prevent multiple low-dose STZ induced diabetes development, suggesting that the same miRNA may play different roles in different organs and conditions. Loss of total miRNAs in hematopoiesis prevents STZ-induced diabetes indicating that essential regulatory role of miRNAs in the process of immune cell mediated islets inflammation.3, HDAC3knockout affects β cell proliferation and function, unlike the protective role of HDAC inhibitors during cytokine mediated islets inflammation, loss of HDAC3in β cells enhances the sensitivity to STZ induced diabetes. HDAC3silencing in vitro reduces the mRNA level of insulin as well as insulin content. Upregulation of SOCS3as a result of HDAC3silence possibly involves in this process.