Mir-375 Mediates Leptin Signal In Affecting POMC Expression Of Hypothalamus

The hypothalamus is an important region of the central nervous system that controls appetite,body weight and metabolism.It consists of multiple types of neurons that fine-tune the expression of specific genes to sense hormones and nutritional signals,integrating and generating appropriate responses.While,the mechanism of how the neuronal genes such as POMC,LEPRB and AgRP regulate the nutrient metabolism is not completely researched.As a key regulator of gene expression,microRNA-375(miR-375)has been frequently researched in metabolic system studies recent years.In this study,the mouse model through third ventricle injection,in situ hybridization,immunofluorescence,Real-time PCR and Western blot was performed to research the mechanism of miR-375-mediated leptin signal regulating POMC expression in hypothalamus.The following results were obtained:1.Expression of miR-375 in LEPRB and POMC neurons of hypothalamus.The expression and distribution of miR-375 in the hypothalamus of mice were detected by frozen section and in-situ hybridization.The results show that miR-375 was widely expressed in the hypothalamus,especially in the arcuate nucleus region.Furthermore,in-situ hybridization and immunofluorescence staining confirmed that miR-375 was expressed in LEPRB and POMC neurons in the arcuate nucleus of mice hypothalamus,with the expression ratio of 52.9%and 81%,respectively.2.Leptin signal can regulate the expression of miR-375 and POMC in hypothalamus.Firstly,leptin was injected into the third ventricle to explore the effect of leptin on the expression of miR-375 and POMC.The results show that after leptin injection,the expression of miR-375 in hypothalamus decreased by 30.3%,while the mRNA and protein expression of POMC increased by 35.4%and 71.6%,respectively.Then,leptin level was increased in obese mice induced by high fat diet,and the expression changes of miR-375 and POMC in hypothalamus were detected.The results show that the expression of miR-375 in hypothalamus of mice fed with high fat diet was reduced by 22.1%.The mRNA and protein levels of POMC increased by 18.8%and 38.2%,respectively.Finally,leptin levels were decreased by fasting mice for 12 h,and then the expression changes of miR-375 and POMC in hypothalamus were detected.It was found that the level of miR-375 in hypothalamus increased by 219.8%,while the mRNA and protein levels of POMC decreased by 49.9%and 56.2%,respectively.These results suggest that leptin may inhibit the expression of miR-375 in hypothalamus,while it can promote the expression of POMC.3.Leptin regulates POMC expression in hypothalamus through miR-375.The miR-375 knockout(KO)mouse model constructed earlier in our laboratory was performed for this study,the genotype of KO mice was identified by PCR,and the body weight of mice were recorded continuously for 8 weeks from weaning.The results showed no significant difference in the body weight between KO mice and the control group wild(WT)mice.After leptin injected of KO mice,the mRNA and protein levels of POMC in hypothalamus were increased by 65%and 130.6%,respectively,and increased 29.6%and 59%compared with WT mice injected with leptin,respectively.Compared with KO mice in normal diet,after 8 weeks of high fat diet,there was no significant change in fat mass and body weight of KO mice,and the mRNA and protein levels of POMC in hypothalamus increased by 58.4%and 98.9%,respectively.Compared with WT mice fed high-fat diet,POMC mRNA and protein levels in hypothalamus were up-regulated by 39.6%and 60.7%,respectively.Compared with nonfasted KO mice,the mRNA and protein levels of POMC in hypothalamus of KO mice after 12 h fasting were decreased by 26.3%and 17.1%,respectively,while the mRNA and protein levels of POMC in hypothalamus of KO mice were increased by 23.6%and 39.1%,respectively,compared with fasting WT mice.These results suggest that leptin may regulate POMC expression in hypothalamus through miR-375,thereby affecting metabolism and weight gain in mice.4.miR-375 may regulate POMC expression through the STAT3 signaling pathway.The hypothalamic leptin regulates POMC expression mainly via the STAT3 signaling pathway.Therefore,we detected the activation of STAT3 signaling pathway in each group of mice,and the results showed that the phosphorylated STAT3(pSTAT3)level increased 159.3%after injection of leptin of mice.The pSTAT3 level in hypothalamic of obese mice increased by 105.7%.After fasting for 12 h,pSTAT3 level in hypothalamus decreased by 57.5%.After leptin was injected of KO mice,the level of pSTAT3 in hypothalamus increased by 246.8%,compared with 87.5%in WT mice with the same treatment.After 8 weeks of high fat diet in KO mice,the hypothalamic pSTAT3 level increased by 128.4%,which was 22.7%higher than that in obese WT mice.There was no significant change in hypothalamic pSTAT3 in KO mice after fasting for 12 h.The above experimental results proved that the hypothalamic neurons of mice expressed miR-375,and leptin may up-regulate the level of pSTAT3 by inhibiting the expression of miR375,and then regulated the expression of POMC,ultimately affecting the metabolism and weight gain of mice.The results have certain guiding meaning for the future treatment of obesity and diabetes.