The Transcriptional Regulation And Functional Research Of Fat Mass And Obesity Associated Gene FTO

Obesity is one of the most prevalent metabolic disease in the world. It is the killer of the public heath owing to its association with insulin resistance, type2diabetes, cardiovascular diseases and some of cancers. FTO is an obesity candidate gene associating with BMI and obesity by GWAS.In this study, we cloned the promoter and CDS sequence of porcine FTO. We analysed the promoter activity and the binding sites of transcription factors to porcine FTO promoter by the dual-luciferase reporter assay system and mutating the binding sites of transcription factors.We also studied the function of FTO by real-time quantitative PCR, triglyceride and glyceride assay, mitoTracker7514staining and flow cytometry, et al. And we got following results:1. Primer sequences of FTO promoter were designed according to published sequence of porcine FTO5’-flanking sequence in NCBI.We predicted the biding sites to FTO promoter by the website of Genomatix. The activity of FTO promoter regulated by the transcriptional factors of PPARγ、CEBPα、CREB2、ChREBP,KLF6and KLF15was detected by the dual-luciferase reporter assay system in NIH/3T3cell line. The experiment result showed that the promoter activity was significantly upregulated and downregulated by KLF6and KLF15respectively compared with the control vector pcDNA3.1.2. FTO mRNA expression in the HEK293A cell in which KLF6and KLF15were overexpresssed respectively was detected by qRT-PCR. The data showed the FTO mRNA level was increased by KLF6and reduced by KLF15.3. In order to confirm whether KLF6and KLF15bind the FTO promoter directly, we mutated the binding sites predicted in the FTO promoter. The activity of the KLF6and KLF15binding sites mutant promoters were recovered in NIH/3T3cell line when co-transfected KLF6and KLF15respectively compared with the wild-type promoter. That suggested KLF6and KLF15possibly regulate the activity of FTO promoter through binding to the predicted binding sites.4. We found that FTO enhanced lipid deposition of hepG2cell by triglyceride assay kit. We demonstrated intracellar TG content was increased by FTO owing to enhancing intracellar TG synthesis and inhibiting TG hydrolization by detecting mRNA levels of fatty acid synthesis and oxidation genes and glyceride assay kit. We also found FTO could significantly upregulate the expression of PLIN and ADRP of lipid droplet associated genes by qRT-PCR.5. We overexpressed FTO in HEK293A cell and detected mtDNA by qRT-PCR and fluorescence intensity by flow cytometry. The result demonstrated FTO could significantly downregulate intracellar mitochondrial mass. We studied mRNA levels of genes which regulated mitochondrial fusion, fission and synthesis by qRT-PCR. We confirmed that mitochondrial DNA decreased by FTO because of increasing mitochondrial fusion and inhibiting mitochondrial fission.