LncRNA-p3134 Interferes Cell Differentiation And Insulin Sensitivity Of 3T3-L1 Pre-adipocyte By Regulating PASK-PPARγ Signaling

BackgroundAdipose tissue is one of important targets of insulin in human body.Under the regulation of insulin,fat cells maintain energy homeostasis in the body by storing and releasing energy through the synthesis and decomposition of triglyceride.The pathological changes of obesity are manifested by the expansion of adipocytes or the increase in the number of adipocytes caused by over-differentiation of mesenchymal stem cells scattered around adipose tissue.Therefore,to explore the relevant changes during the period that mesenchymal stem cells differentiate into mature adipocytes,to find transcription factors involved in the regulation of mesenchymal stem cell differentiation,and to complete the molecular mechanisms of lipid metabolism and obesity genesis,are of great significance in providing new targets in clinical treatment of obesity and lipodystrophy.Long non-coding RNA is widely involved in various physiological and pathological processes in humans through transcriptional regulation and post-transcriptional modification.In recent years,it has been reported that differently expressed LncRNAs are found in tumors,metabolic diseases,and neurodegenerative diseases.Among them,LncRNA-p3134 is closely related to obesity and type 2 diabetes.It is a potential predictor and intervention target for obesity,Functional changes were observed in mouse islets and 3T3-L1 cell lines with LncRNA-p3134 overexpression,but the potential binding site and mechanism is still poorly understood.This study was to explore the effect of LncRNA-p3134 on the function and differentiation of transgenic pre-adipocytes and its possible mechanism.ObjectiveThe differentiation function and insulin sensitivity of mice 3T3-L1 cell line overexpressing LncRNA-p3134 were compared with those in control group.The effect of LncRNA-p3134 on the differentiation and function of transgenic adipocytes were investigated.The potential sites and mechanisms by which LncRNA-p3134 produces regulation were then analyzed.Methods(1)Lentiviral transfection technology was used to construct a 3T3-L1 cell line with stably expressing LncRNA-p3134,after inducing differentiation of pre-adipocytes into mature adipocytes,oil red O staining and semi-quantitative method were used to observe the impact of LncRNA-p3134 on lipid droplet size and morphology.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was applied in detection of the relative expression levels of adipogenic genes such as PPARγ,C/EBPα,FABP4,as well as adipokines such as Leptin and Adiponectin.Western blotting.(Western blot)technique were applied to detect the expression of related proteins.Insulin-stimulated glucose uptake experiments were performed on well differentiated mature 3T3-L1 cells.The glucose concentration in the medium before and after insulin stimulation was determined by colorimetry,and the uptake rate of glucose under insulin stimulation was then calculated.(2)According to the sequence information of lncRNA-p3134,sequence fragments share high similarity were matched in the mouse gene database to predict the potential combination site of LncRNA-p3134.SiRNAs were then transfected into 3T3-L1 adipose cell precursor to construct a cell line with the knockout of the gene of interest.In the cell model overexpressing LncRNA-p3134 or target gene knocked out,PPARγ was activated by rosiglitazone treatment,and adipocytes differentiation after rosiglitazone treatment were detected by detecting the size and quantity of lipid droplets with the application of oil red O staining and semi-quantitative method.Gene and protein expression levels of related markers were detected by RT-PCR and Western blot.The glucose uptake rate of adipocytes stimulated by insulin was detected by colorimetry.ResultsCompared with 3T3-L1 cells in control group,the cells overexpressing LncRNA-p3134 showed a significant decrease in lipid droplets after induction of differentiation,and the expression levels of adipogenetic markers such as PPARy,FABP4 and C/EBPa were significantly decreased,and the.Decreased glucose uptake rate were observed in cells overexpressing LncRNA-p3134 after insulin stimulation,suggesting that LncRNA-p3134 reduces the differentiation viability of pre-adipocytes.Sequence analysis revealed that LncRNA-p3134 and the PASK mRNA share a matching segment with a length of 37bp located at the 10th exon of PASK mRNA,suggesting that LncRNA-p3134 may conduct its regulatory effect via PASK.qPCR results showed that LncRNA-p3134 overexpression significantly reduced the transcriptional expression of PASK.PASK in 3T3-L1 cells were knocked down by siRNA,and the results showed that the expression of PPARy was decreased in the PASK knockdown cell line,with pre-adipocyte differentiation process and function interfered.This is in consistency with the phenotype of 3T3-L1 cell overexpressing LncRNA-p3134.Treatment of rosiglitazone in LNCRNA-p3134 overexpressing 3T3-L1 cell line and PASK knockdown cell line increase the glucose uptake rate after stimulated by insulin.ConclusionsLncRNA-p3134 inhibits the differentiation of mouse 3T3-L1 pre-adipocytes into mature adipocytes,interferes adipocyte function such as adipokine secretion and sensitivity to insulin.Regulatory mechanism of LncRNA-p3134 might be associated with PASK protein.PASK expression was negatively regulated by LncRNA-p3134 overexpression.The differentiation of pre-adipocytes into mature adipocytes is regulated by LncRNA-p3134 or PASK via PPARy.Rosiglitazone acted as a PPARy activator,partially rescued adipocyte functioning in condition of LncRNA-p3134 overexpression and PASK knockdown.It is suggested that LncRNA-p3134 inhibits the differentiation of adipose stem cells and is a potential intervention target for obesity.