.1. Of P38 On Cox-2 Transcriptional Regulation Of The Role Of Pancreatic ¦Â Cell Function Analysis Of Transcription Of The Luciferase Reporter Gene Regulatory Element Method Study

Type 1 diabetes is primarily to autoimmune-mediated destruction of pancreaticβ-cells,resulting in absolute insulin deficiency.The underlying cause of this serious chronic illness is not well understood.Infiltration of islets of Langerhans by immune cells is likely to be an initial event in development of type 1 diabetes.Invading macrophages and T-cells may secrete proinflammatory cytokines promoting the destruction of insulin-producingβ-cells.Therefore,research aimed at characterizing the mechanism of the dysfunction in pancreaticβ-cell are critical important both for understanding the pathogenesis of type 1 diabetes and improving outcomes in islet transplantation.Cyclooxygenase-2(COX-2)is a key enzyme that catalyzes the conversion of arachidonic acid to prostaglandins.It plays an important role in inflammation,carcinogenesis,and the development of diabetes.Although COX-2 gene regulation is deeply and widely investigated,some important mechanism underlying its post-transcriptional regulation is still unknown.To investigate the role of p38 pathway in PGE2 formation and that could be involved in cytokine-mediated dysfunction of pancreaticβ-cells,the present study was designed to identify the precise mechanism of p38 pathway in COX-2 expression and PGE2 formation using IL-1βand p38 inhibitor SB203580 in HIT-T15 cells.We found that in HIT-T15 cells,IL-1βcan enhanced the stability of COX-2 mRNA through p38 pathway,thus leading to up-regulation of COX-2 expression and promoting the destruction of insulin-producingβ-cells.It is likely that IL-1βenhanced the stability of COX-2 mRNA by inducing the cytoplasmic shuttling of HuR.Luciferase reporter genes have been widely used for the functional characterization of regulatory elements in the 3'UTR.In our research,using transient expression assay system with Rin m5F cell or NIT-1 cell lines,we first demonstrated that luciferase reporter gene constructs reproducibly show not only the elements with special sequences in the 3'UTR can affect the luciferase activity,but DNA fragments with the random sequences ligated into the same site of the luciferase reporter gene vector can decrease the luciferase activity,and the extent of the decrease is dependent on the length of the DNA fragments.Our findings strongly suggested a need for re-examination of 3'UTR characterizations of many eukaryotic genes,which have been studied to date with luciferase reporter genes.