| Objective: Obesity is a high risk factor for type 2 diabetes mellitus,hypertension and cardiovascular and cerebrovascular diseases.The research on the pathogenesis of obesity has important clinical significance.The increase in the number or size of fat cells caused by abnormal differentiation of fat cells will lead to the increase of adipose tissue and lead to obesity.Therefore,it is of great significance to study the regulation mechanism of adipocyte differentiation for the pathogenesis of obesity.Based on the establishment of human adipocyte differentiation model,this study studied the function of lnc RNAHOXA11-AS1 in adipocyte differentiation and the regulation of FABP4 protein expression at post-transcriptional and translational levels by interacting with Hu R.To reveal the mechanism of lnc RNA HOXA11-AS1 promoting adipocyte differentiation,and provide important theoretical basis for the understanding,prevention and treatment of obesity in the future.Methods: 1)Abdominal omental adipose tissue samples and blood samples were collected from obese and non-obese groups,and m RNA expression levels of lnc RNA HOXA11-AS1,FABP4,CEBP-α,FASN and ACC were detected by RT-q PCR.Using the separated serum samples,fasting blood glucose,triglyceride,high density lipoprotein,low density lipoprotein and other clinical biochemical indexes were detected in the two groups.2)PROMO software predicted the binding site of transcription factor PPARγ in lnc RNAHOXA11-AS1 promoter region,and Ch IP assay and biluciferase reporter assay were used to determine the transcriptional regulation of PPARγ on lnc RNAHOXA11-AS1.3)lnc RNAHOXA11-AS1 m RNA expression was overexpressed and knocked down.Lipid droppings formation and morphological changes of adipocytes were detected by oil red O staining,and mrna and protein expression levels of adipocyte differentiation-related genes CEBP-α,FABP4,FASN and ACC were also detected.4)The binding of Hu R to lnc RNA HOXA11-AS1 and FABP4 was confirmed by RNA-IP experiment,and the binding of lnc RNA HOXA11-AS1 to Hu R was further verified by RNA pull-down experiment.5)The effects of lnc RNA HOXA11-AS1 on nuclear and mass migration in Hu R were detected by nuclear and plasma isolation and immunofluorescence assay.6)After overexpression and knockdown of Hu R m RNA and protein expression,the changes of FABP4 m RNA and protein expression levels were detected by RT-q PCR and western-blot,and the influence of Hu R on the stability of FABP4 m RNA was detected by actinomycin D assay.7)The software predicted the possible Hu R binding site sequence(ARE element)on FABP4 m RNA,and the binding site of Hu R and FABP4 was determined by double luciferase reporter gene experiment.Results: 1)Analysis results of clinical samples showed that m RNA expression levels of lnc RNA HOXA11-AS1,FABP4,CEBP-α and FASN in obese patients were significantly increased compared with non-obese patients,with statistical significance(P < 0.05).lnc RNA HOXA11-AS1 m RNA expression level was positively correlated with hip circumference,and was positively correlated with FABP4,CEBP-α,FASN and ACC m RNA expression levels.2)PROMO software predicted that lnc RNAHOXA11-AS1 promoter region had binding sites for transcription factor PPARγ.Ch IP results showed that PPARγ could bind to the lnc RNAHOXA11-AS1 promoter 48 h after adipocyte differentiation.Biluciferase reporter gene assay showed that PPARγ could increase lnc RNA HOXA11-AS1 transcription.3)After the expression of lnc RNA HOXA11-AS1 was knocked down,the formation of lipid droplets in adipocytes was reduced,and the m RNA and protein expression levels of FABP4,CEBP-α,FASN and ACC were significantly decreased,with statistical significance(P < 0.05).However,after lnc RNA HOXA11-AS1 overexpression,the formation of lipid droplets in adipocytes increased,and the m RNA and protein expression levels of FABP4,CEBP-α,FASN and ACC were significantly increased,with statistical significance(P < 0.05).After lnc RNA HOXA11-AS1 was knocked down or overexpressed,the protein expression level of FABP4 changed most significantly,with statistical significance(P < 0.01).4)Starbase software predicted that lnc RNA HOXA11-AS1 could bind to RNA binding protein Hu R.Rna-ip and RNA pull down experiments confirmed that Hu R could bind lnc RNA HOXA11-AS1 and FABP4 m RNA,respectively,and the binding between Hu R and FABP4 was significantly reduced after the expression of lnc RNA HOXA11-AS1 was knocked down.The difference was statistically significant(P < 0.05).5)The results of immunofluorescence and nucleoplasmic separation showed that Hu R migrated from nucleus to cytoplasm during adipocyte differentiation.However,after the knockdown of lnc RNA HOXA11-AS1 expression,the amount of Hu R protein migrating from the nucleus to the cytoplasm was decreased,with statistical significance(P < 0.05).6)FABP4m RNA expression level remained unchanged in overexpression and knockdown Hu R,while protein expression level was significantly increased and decreased,with statistical significance(P < 0.05).The results of line D experiment showed that the half-life of FABP4 m RNA became shorter and its stability decreased after Hu R knockdown.7)The results of dual luciferase reporter gene experiment showed that Hu R could bind FABP43’UTR ARE elements,and the binding of FABP4 3’UTR ARE1 mutation significantly decreased,with statistical significance(P < 0.05).Conclusions: 1)lnc RNAHOXA11-AS1 was specifically highly expressed in adipose tissue of obese patients,and the m RNA expression level of lnc RNA Hoxa11-AS1 in adipose tissue was positively correlated with the mrna expression levels of adipose-differentiation related genes FABP4,CEBP-α,FASN and ACC.2)lnc RNAHOXA11-AS1 is transcriptionally regulated by PPARγ.By binding to Hu R,lncrnahoxa11-AS1 can promote the transfer of Hu R from nucleus to cytoplasm,increase the binding of Hu R to FABP4,and thus increase the stability of FABP4 m RNA,up-regulate the protein expression level of FABP4,and increase the formation of lipid droplets in adipocytes.Finally promote adipocyte differentiation. |
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