The Mechanism Of LncRNA-p3134 In Regulating Pancreatic Beta Cell Function And Insulin Signaling Pathway

BackgroundIslet is an important endocrine organ that regulates glucose metabolism and maintains blood glucose homeostasis through the secretion of insulin.The genetic background of increasing risk of diabetes and the environmental factors will eventually lead to the dysfunction of islet ? cells.Therefore,it is of great significance to explore the susceptible gene of diabetes,and to understand its role and pathogenesis in the development of diabetes.Long non-coding RNA(1ncRNA)extensively participates in almost every pathophysiological process and regulates gene expression at various levels,including epigenetic,transcription and post-transcription level.Recent studies have indicated that the abnormal expression of 1ncRNAs is closely related to the development of tumor,cardiovascular disease,infection,nervous system diseases and diabetes mellitus,which can promote or inhibit the occurrence of disease.The aim of this study was to explore the relationship between lncRNA-p3134 and the glucose metabolism of islet beta cells as well as insulin signaling pathway.ObjectiveTo screen the differential expression of circulating IncRNA in patients with type 2 diabetes mellitus and normal people,and to explore the effect of lncRNA-p3134 on islet ? cell function and the possible mechanism of participating in the development of type 2 diabetes mellitus.MethodsLncRNA microarray technology was used to identify the differentially expressed circulating IncRNAs in T2D patients.RT-PCR analyses were performed to determine the expression of lncRNA-p3134 in 30 pairs of diabetic and non-diabetic patients.The correlation of lncRNA-p3134 to clinical data from T2D patients was analyzed.The expression of lncRNA-p3134 in serum exosome was also detected.Additionally,lncRNA-p3134 was overexpressed in Min6 cells by adenovirus-mediated technology and then determined its expression in different glucose concentration.CCK-8,flow cytometry and TUNEL were performed to determine the effect of lncRNA-p3134 on proliferation and apoptosis.Western blot was used to determine the expression of apoptosis related protein.Glucose-stimulated insulin secretion(GSIS)was performed to detected the insulin secretion.The expression of insulin-related transcription factors including Pdx-1?MafA?GLUT2?TCF7L2 was also detected by RT-PCR and Western blot.Then we observe whether high glucose-induced Min6 cell dysfunction was reversed by overexpression of lncRNA-p3134.In vivo,lncRNA-p3134 was overexpressed in db/db diabetic mice by intraperitoneal transfection via tail vein.Intraperitoneal glucose tolerance test(IPGTT)was performed and the level of insulin release was measured.The expression of insulin-related transcription factors was detected by RT-PCR and the proportion of insulin-positive cells in pancreatic tissues was observed by immunohistochemistry.The expression of PI3K,Akt and mTOR proteins was detected by Western blot after lncRNA-p3134 overexpression in both vivo and vitro.GSIS function was analyzed after using PI3K pathway inhibitor(LY294002).ResultsThe circulating level of lncRNA-p3134 was higher in diabetic patients than in non-diabetic controls and was positively correlated with fasting blood glucose and negatively correlated with HOMA-? levels(r=-0.412,p<0.05).The lncRNA-p3134 had risen by 4 times in serum exosomes but nearly unchanged in exosome-free samples.Bioinformatics analysis indicated IncRNA-p3134 was relevant to insulin signaling pathway and TCF7L2 regualtion.The secretion of IncRNA-p3134 was dynamically modulated by glucose in Min6 cells.LncRNA-p3134 was upregulated in moderate high glucose level but significantly decreased in glucotoxicity concentration.LncRNA-p3134 positively regulate GSIS through promoting of key regulators(Pdx-1,MafA,GLUT2 and TCF712)in ? cells.In addition,the overexpression of IncRNA-p3134 resulted in a decreased apoptosis ratio and partially reversed the glucotoxicity effects on GSIS function in Min6 cells.In vivo,IncRNA-p3134 overexpressed in db/db mice improved glucose tolerance and insulin secretion after IPGTT.The increase of the transcription factors and the insulin positive cells areas by upregulation of lncRNA-p3134 in db/db mice confirmed the compensatory role of lncRNA-p3134 to preserve ?-cell function.Furthermore,the protective effect of IncRNA-p3134 on GSIS by positive modulation of PI3K/Akt/mTOR signaling was also confirmed.After blocking the PI3K/AKT signals with their specific inhibitor,the effect of overexpressed lncRNA-p3134 on insulin secretion was obviously attenuated.Conclusions:LncRNA-p3134 which is characteristically expressed in type 2 diabetes mellitus may hve a protective effect on beta cell function by raising the expression of key transcription factors and promoting insulin synthesis and secretion,as a result,delaying the progression of diabetes.This protective effect may be closely related to activating insulin signaling pathway PI3K/Akt/mTOR,which provide a new perspective for further clarifying the regulatory mechanism of islet ? cell function.