The Effect And Mechanism Of LncRNA GM13133 On The Browning Of White Adipocytes

Obesity a pathological state that was caused by the imbalance in the energy metabolism,it is great harm to human body.The core reason of obesity is the body will return excess energy as fat stored in white fat cells and lead to abnormal accumulation and distribution of white adipose tissue in the body.So how to healthy,effective reduce the content of white adipose tissue is the key to treat obesity problem.There is also a kind of previously neglected in the human body adipose tissue-brown adipose tissue,due to the thermal power and healthy way of"combustion" in fat and become the ideal target treatmentof obesity.White fat under certain conditions also has the characteristics of the brown fat,and this phenomenon known as white fat browning or beigeing.Long chain noncoding RNA(lncRNA),is akind of noncoding RNA molecules that itself is not encoded proteins,the length of transcription more than 200nt and that make up most of the human genome noncoding transcript,play an important role in life activities.The role of forms was various,and it is closely related to X-chromosome inactivation,genomic imprinting,chromatin modification and transcription activation and inhibition,RNA shear,etc.LncRNAs whether play a regulating effect in white fat browning.At present did not see the related reports.In order to screen for lncRNAs that may be involved in the brownish effect of white fat,we used gene chip technology to compare the differences in lncRNAs between white fat and brown adipose tissue and found Chain non-coding was high expression in brown adipose tissue and low expression in white adipose tissue called GM13133.Through the bioinformatics analysis of the basic characteristics and prediction of potential function about GM13133 found that length was736bp.was located in 4qE2 of chromosome and was in the first intron of PRDM16 gene,and the transcription direction was opposite to PRDM16.UCSC online database analysis found that GM13133 exists in different species with high conservatism.Based on the cis role of lncRNA that regulates the expression of adjacent genes(located in the first intron of PRDM16),we speculate that GM13133 may have the effect of promoting fat energy metabolism--browning.To reveal the potential function of GM13133,we first used the lentivirus overexpression system to establish a stabilize white adipocytes of GM13133 and examine their effect on adipocyte differentiation.The results showed that GM13133 could significantly down-regulate the expression of differentiation-related genes and proteins.The overexpression of GM13133 could decrease the number and size of lipid droplets by oil red O test.The triglyceride test kit was used to detect the the amount of triester and its expression was also significantly reduced.The above indicate that overexpression of GM13133 can inhibit the white fat differentiation,expected to provide a new target for prevention and treatment of human obesity.The study also indicate that the expression of UCP-1 and PGC1-? increased significantly,the number of mitochondrial copy was increased and the oxygen consumption rate was significantly increased,which showed the characteristics of brown adipocyte-like cells,while GM13133 overexpressed Does not affect the proliferation of white fat cells.We further explored mechanism of GM13133 to promote the browning,found that overexpression of GM13133 did not change the content of PRDM16,so we guess that GM13133 may be through the cAMP signal pathway to regulate PRDM16 activity to play a function.Further revealing the molecular mechanism of the conversion of white fat to brown fat,looking for new molecules or new drugs that promote the conversion of white fat to brown fat,which may bring bright prospects for obesity treatment.Part ? The found and basic biological characteristics of GM13133Objective:To observe the expression of LncRNAGM13133 in white fat and brown fat in mice by conservative and homologous biological analysis of GM13133.To explore the correlation between GM13133 with white adipocyte differentiation and brownation,and to provide a theoretical and experimental basis for elucidating GM13133 in adipose tissue.Methods:The geno type of GM13133 was analyzed by UCSC.The inguinal adipose tissue of mouse was selected and the brown adipose tissue of mice was selected.The expression level of GM13133 in white adipocytes and brown adipose tissue was detected by Realtime PCR;cold,beta adrenergic agonists and cAMP stimulated mice white adipose tissue,to detect the expression of GM13133 after stimulationResults:1)GM13133 had better conservation of species,and the target gene function was mainly enriched in the pathway of brown adipose themogenesis and adipocyte differentiation;2)GM13133 was highly expressed in brown adipose tissue;3)In vivolevels of cold,?-adrenal agonist stimulation can promote GM13133 expression in white fat;4)Cell levels CL-316,243 and cAMP treatment induced differentiation of mature adipocytes that could promote GM13133 expression.Conclusion:GM13133 is closely related to white fat differentiation and browning by detecting GM13133 expression in different tissues of mice.It is high expressed inbrown adipose tissue.Brown-regulating factors(cold stimulation,?-adrenergic receptor agonists)can promote the expression of GM13133 in white adipose tissue and cell,the body may play a role in regulating the expression of GM13133 by promoting brown-like regulation.This has laid a theoretical and experimental basis for the subsequent study of GM13133 in the study of the function and mechanism of white fat brown.Part ? GM13133 inhibits white fat differentiation function and analysisObjective:To investigate the effect of GM13133 on inguinals white fat adipogenic differentiation of mouse by overexpression of GM13133 in mouse white preadipocytes cells.Methods:Using the lentiviral vector overexpressed by GM13133,the subcutaneous white adipose precursor cells derived from the groin of mice were infected and the same cells transfected with empty virus were used as the control group.Insulin,dexamethasone,rosiglitazone,1-methyl-3(MIX)was used to induce cell differentiation and maturation.Oil red O was used to observe the formation of lipid droplets during the process of adipogenic differentiation.The content of triglyceride in mature adipocytes was detected by enzyme colorimetry.Real-time PCR and Western Blot were used to detect the key genes of adipocyte differentiation(PPARy,C/EBP?,FABP4 and GLUT4)Results:l)overexpression of GM13133 could decrease the droplets of white fat cells;2)The triglyceride test kit showed that the triglyceride synthesis and GPDH activity were significantly decreased;3)Real-time PCR and Western Blot showed that the gene expression of PPARy2,C/EBP-a,FABP4 and GLUT4 and their protein levels were significantly down-regulated in adipocyte differentiation.Conclusion:Overexpression of GM13133 can inhibit the expression of lipid droplets and triglycerides in white adipocytes.The expression of genes and proteins related to adipogenic differentiation of white fat in mice is down-regulated.GM13133 can inhibit the differentiation of white adipocytes.Part ? GM13133 promotes white fat browning function and analysisObjective:To explore the function and mechanism of induce browning and energy metabolism after GM13133 overexpression.Methods:Using the lentiviral vector overexpressed by GM13133,the subcutaneous white adipose precursor cells derived from the groin of mice were infected and the same cells transfected with empty virus were used as the control group.insulin,dexamethasone,rosiglitazone,1-methyl-3-isobutyl induce preadiposityes into mature adiposityes.The difference of mitochondrial copy number between control group and overexpression group was detected by PCR and electron microscopy.The difference of UCP-1 was detected by immunofluorescence.The difference of oxygenconsumption rate by seahorse test and Realtime PCR and Western Blot was used to detect the expression of brown adipose-specific genes or brown-related genes.Bioinformatics analysis the locations of GM13133,real-time quantitative PCR and Western Blot to predict its mechanism.RNA-seq technique was used to analyze the difference between overexpression GM13133 and control group mRNA.Results:1)Realtime PCR and electron microscopy showed that the expression of mitochondrial copy number in GM13133 group was significantly up-regulated;2)Immunofluorescence assay showed that the expression of UCP-1 in GM13133 group was significantly higher than that in control group;3)By real-time quantitative PCR and Western Blot detect of white adipocytes brown key genes and protein levels reveal that the level in overexpression of GM13133 was significantly up-regulated;5)by bioinformatics analysis and PCR Western blot analysis showed that the downstream target of GM13133 was PRDM16,but there was no significant difference between overexpression group and control group,and the expression of cAMP was significantly higher.6)RNA-seq analysis showed that there were 523 genes in the control group and the GM13133 group,and the P value was less than 0.05,including 217 up-regulated genes and 306 down-regulated genes,in which the cAMP signaling pathway was different Most obvious.Conclusion:1)GM13133 up-regulated the expression of white adipose-related genes,brown-related genes and proteins;2)GM13133 could bind to the target gene-PRDM16 intron but out affect its expression level;3)The themogenesis signal pathways about overexpression of GM13133 that may be rely on cAMP signaling pathways.The results show that GM13133 can promote white fat browning and energy consumption.Its mechanism may be related to the cAMP signal pathway.