The Expression Profile Of Circular RNA H19 In Metabolic Syndrome And Its Role In Adipogenic Differentiation

Background and Aim:The metabolic syndrome(Met S)is characterized of a cluster of medical disorders including central obesity,hypertension,hyperlipemia and insulin resistance.Insulin resistance caused by abnormal lipid metabolism represents a key driver for Met S.Lipid metabolism includes lipid synthesis and decomposition,which is related to the process of adipogenic differentiation.The lipid metabolism disorder caused by Met S will lead to the impairment of the adipogenic differentiation of h ADSCs.In recent years,studies have shown that circular RNA(circ RNA)is closely related to the occurrence of metabolic diseases.In this study,we aim to explore the expression profile of human circular RNA H19(hsa＿circ H19)in Met S and its role in adipogenic differentiation.Methods:The blood samples from Met S patients and non-Met S subjects were used to determine the expression level of the hsa＿circ H19.Spearman correlation analysis was used for analysis between hsa＿circ H19 and metabolic parameters.Multivariate analysis was used to analyse the influence of hsa＿circ H19 on Met S.To further investigate the correlation between hsa＿circ H19 and adipogenic differentiation,h ADSCs were selected for in vitro experiments.Using specific divergent primers amplification and RNase R experiment,hsa＿circ H19 was identified to generate from H19 by back-splicing.After knock-down of hsa＿circ H19 in h ADSCs,q RT-PCR and Western Blotting were used to evaluate the expression level of adipogenic genes including PPARγ,C/EBPα,LPL and SREBP1.The changes of SREBP 1protein in nucleus and cytoplasm were further detected by protein nucleo-cytoplasmic separation assay.Oil red O,Nile red staining assay and triglyceride assessment were performed to examine the role of hsa＿circ H19 in h ADSCs differentiation.Then,bioinformatics analysis,RNA Pull-down and RIP assays were conducted to explore the related RNA binding protein of hsa＿circ H19.Immunofluorescence assay was performed to determine the SREBP1 protein localization and translocation.Finally,functional complementation analysis was used to determine the function of hsa＿circ H19 in regulating SREBP1 protein translocation in the presence of PTBP1.Results:The expression of hsa＿circ H19 in serum samples of Met S patients was increased obviously compared to non-Met S patients(p<0.001).The level of circulating hsa＿circ H19 displayed some degree of correlation with indexes of lipid metabolism,like BMI、WC、WHR、Fat%、TG and VFA.After accounting for confounding factors,high levels of hsa＿circ H19 remained an independent risk factor for Met S.Furthermore,in vitro,the knockdown of hsa＿circ H19 significantly increased the expression of adipogenic genes including PPARγ,C/EBPα,LPL and SREBP1 and the formation of lipid droplets.Bioinformatics analyses revealed that hsa＿circ H19 shared multiple binding sites with polypyrimidine tract-binding protein 1(PTBP1)and their interaction was validated by circ RNA pull-down and RIP assays.Mechanistically,immunofluorescence assay and functional complementation analysis showed depletion of hsa＿circ H19 triggered translocation of sterol-regulatory element binding proteins(SREBP1)from cytoplasm to nucleus in the presence of PTBP1.Conclusion:In consequence,hsa＿circ H19 is highly expressed in Met S,and its expression level is positively correlated with WC,VFA and other lipid metabolism-related factors,which is an independent risk factor for Met S.Furthermore,experimental results showed that knockdown of hsa＿circ H19 promotes h ADCSs adipogenic differentiation via targeting of PTBP1 in vitro.