Effect Of Long Non-coding RNA XIST On Adipose Tissue And Its Mechanism

BackgroundAlthough the incidence of obesity is increasing and obesity is associated with metabolic diseases,most of the existing drugs for treating obesity have poor efficacy or safety.Therefore,new intervention targets for obesity are needed.The activation of brown adipose tissue can improve metabolism in mice and humans,so the research of increasing the activity of brown adipose tissue and promoting the beige coloration of white fat is of great significance for the treatment of obesity.LncRNAs plays an important role in adipocyte differentiation and gene regulation.At present,the effect of lncNRA XIST on adipose tissue and its effect on adipocyte differentiation is unclear.ObjectiveThe purpose of our research is to initially evaluate the effect of lncRNA XIST on adipose tissue,and to explore its effect on brown adipocytes differentiation and its possible mechanism.Method1 Expression of lncRNA XIST in human perirenal and subcutaneous adipose tissuePeripheral adipose tissue and subcutaneous adipose tissue were collected from female without adrenal adenoma control group(n=5),male without adrenal adenoma control group(n=6),and female adrenal cortisol-producing adenoma patients(n=7).Adipose tissue total RNA was extracted.After reverse transcription into cDNA,the expression of lncRNA XIST mRNA in the above tissues was detected by RT-PCR.2 Changes of lncRNA XIST expression before and after mouse adipocytes differentiationThe total RNA of mouse white preadipocytes(3T3-L1)and mouse brown preadipocytes(WT-1)cells before and after differentiation were extracted and reversetranscribed into cDNA.The expression of lncRNA XIST was detected by RT-PCR.The WT-1 cells were collected before and after differentiation,and RNA was extracted after nuclear separation experiments,and the expression of lncRNA XIST was detected by RT-PCR.3 Effect of lncRNA XIST on the differentiation of mouse brown preadipocytes and its mechanismIn vitro culture of mouse brown preadipocytes WT-1,transfected with lncRNA XIST overexpression plasmid(control group transfected with control plasmid)or knocked down lncRNA XIST smart silencer(control group transfected with control sequence)at day-2.And collected cells at day0,day2 and day4 for mRNA and protein expression measure by RT-PCR and Western Blot,respectively.RIP experiments were performed to determine whether lncRNA XIST binds to C/EBPa protein.4 Effects of overexpression of lncRNA XIST on male and female bilateral ovariectomized miceFemale mice were subjected to bilateral ovariectomized(OVX)to make a postmenopausal model,sham operation served as control,and fed with high-fat diet(HFD).Male C57BL6 mice were given either a high-fat diet or a chow diet(CD)according to grouping.The pCMV-XIST plasmid or pCMV-CON plasmid was inj ected by tail vein according to the group.Blood and tissue samples were collected for subsequent testing.Results1 The expression of lncRNA XIST in human adipose tissue has a gender difference.The expression of lncRNA XIST in subcutaneous and perirenal adipose tissue in female CON group was higher than that in male CON group.The expression of XIST in human adipose tissue is different.In women with CPA,the expression of XIST in subcutaneous adipose tissue is higher than that in perirenal fat.In women,the expression of XIST in subcutaneous adipose tissue is lower than in perirenal fat.In CON men group,the expression of XIST in subcutaneous adipose tissue is higher than that of peripranial adipose tissue.2 The expression of XIST decreased significantly in mouse white preadipocytes(3T3-L1)after differentiation,while XIST significantly increased in mouse brown preadipocytes(WT-1)after differentiation.The lncRNA XIST of WT-1 cells were mainly distributed in the nucleus before and after differentiation.3 After overexpression of lncRNA XIST in WT-1 cells,the expression of adipokines PPARγ,C/EBPα,SREBP1C,adiponectin,and UCP1 protein significantly increased,and inhibited SMAD2 phosphorylation and WNT10B protein expression at day2 and day4.After knocking down lncRNA XIST in WT-1 cells,the expressions of adipokines PPARγ,C/EBPα,SREBP1,adiponectin,and UCP1 protein decreased significantly,and promoted SMAD2 phosphorylation and WNT 1 OB protein expression at day2 and day4.RIP experiments confirmed that lncRNA XIST can bind to C/EBPa protein.4 Overexpression of XIST in female mice can improve dyslipidemia and decreased weight gain in HFD+OVR mice,and can improve white fat and brown adipose tissue dysfunction caused by HFD+OVR.Overexpression of XIST in male mice can improve dyslipidemia and decreased weight gain in mice caused by HFD and improve white and brown adipose tissue dysfunction in miceConclusionLncRNA XIST was significantly upregulated in mouse brown preadipocytes after differentiation.In vitro experiments confirmed the promotion of lncRNA XIST on mouse brown preadipocyte differentiation.In vivo experiments through overexpression of lncRNA XIST intervention showed that it can resist high-fat diet-induced obesity and improve adipose tissue function.LncRNA XIST may be a new target for obesity treatment.