Role Of NFIA In Adipocyte Differentiation And The Mechanisms Involved In The Process

Objective: NFIA is a member of nuclear factor I(NFI)transcription factor family,Transcription factors mainly activate or inhibit the transcription of target genes by directly binding to the promoter region of the target genes or recruiting other transcription factors.NFIA is involved in a series of biological processes including regulation of differentiation,development,proliferation and apoptosis of a variety of cells.Studies have shown that the NFI family is involved in bone metabolism and bone homeostasis,but NFIA regulation of mesenchymal stem cells or bone marrow stromal cells to adipocyte differentiation has not been reported.Therefore,this study mainly investigated the effect of transcription factor NFIA on adipocyte differentiation and explored its mechanism.Methods: 1?By using quantitative qRT-PCR,We examined the expression of NFIA in the different tissues of 4-week old C57BL/6J mice and tested the expression of NFIA in the bone marrow stromal cell line ST2 cells after treated with adipogenic medium.2?The NFIA overexpression plasmid or NFIA siRNA were transfected into ST2 or C3H10T1/2 and adipogenic induction was completed when the cell fusion rate reached 100%,The effects of NFIA on adipogenic differentiation were investigated by Oil Red O staining,qRT-PCR and Western Blotting.3?We constructed the knock-down lentiviral plasmid of NFIA and packaged the Nfia-shRNA lentivirus particles with 293 T cells.Mouse BMSCs cells were infected with the lentiviruses.The effects of Nfia-shRNA LV on adipogenesis were detected by Oil-red O staining and qRT-PCR.4?The endogenous expression of NFIA was inhibited by siRNA in ST2 cells.The non-phosphorylation of ?-catenin,a key protein in Wnt signaling pathway,was detected by Western Blotting;After overexpression of Nfia,ST2 cells were treated with Wnt3 a and the effect of NFIA on ?-Catenin nuclear translocation was detected by immunofluorescence assay.The mRNA levels of Lrp5,Lrp6,Sfrp1 and Wnt10 b genes associated with Wnt signaling pathway were detected by using qRT-PCR.5?Bioinformatics was used to predict the binding sites between NFIA and the target gene Sfrp1 promoter regions;The Sfrp1 promoter was cloned into the luciferase reporter vector and Nfia siRNA was co-transfected with the Sfrp1 promoter in ST2 cells.Dual-luciferase reporter assays were performed to determine whether NFIA affects the transcriptional activity of the Sfrp1 promoter.Chromatin coimmunoprecipitation was used to further examine the association of NFIA with the predicted promoter regions of Sfrp1.6?the Sfrp1 siRNA were transfected into ST2 and adipogenic induction was completed,the effects of Sfrp1 siRNA on adipogenic differentiation were investigated by Oil Red O staining,qRT-PCR and Western Blotting;Under the background of Sfrp1 gene silencing,to study whether the effect of NFIA on adipocyte differentiation is blocked.Results: 1?The distribution of NFIA in various tissues of C57 BL / 6J mice showed that NFIA was highly expressed in mouse heart,liver,bone,perirenal fat,inguinal fat,epididymal fat and omental fat.During the induction of ST2,the mRNA expression of Nfia increased,and reached its peak on the 5th day of adipogenic induction.2?Overexpression of NFIA and induction of adipogenesis in ST2 cells or C3H10T1/2 cells showed that NFIA promoted the differentiation into adipocytes,and the expression of Peroxisome proliferator-activated receptor ?(PPAR?),CCAAT enhancer binding protein ?(C/EBP?),Adipocyte fatty acid-binding Protein 2(aP2)and Adipsin were up-regulated.In contrast,Nfia siRNA inhibits its differentiation into adipocytes.3 ? We successfully constructed the lentiviruses of knockdown endogenous NFIA.Nfia-shRNA LV knockdown lentivirus inhibits the expression of endogenous NFIA.Oil red O staining showed that Nfia-shRNA LV knockdown lentivirus inhibited the differentiation of BMSCs into adipocytes,qRT-PCR results showed that the mRNA expression of specific genes related to adipocyte differentiation was decreased.4?NFIA siRNA inhibits the expression of endogenous NFIA in ST2 cells and,and Western Blotting results show that it can increase the level of non-phosphorylated ?-catenin;After overexpression of Nfia,the immunefluor-esceence assay detects the nuclear transport of ?-Catenin is inhibited.qRT-PCR results showed that the mRNA expression of Sfrp1,a negative regulator upstream of wnt signaling pathway,was significantly decreased.5?Double luciferase reporter assays demonstrated that knockdown of NFIA inhibited Sfrp1 transcriptional activity;The results of chromatin immunoprecipitation further confirmed the physical binding of NFIA protein to the promoter of target gene Sfrp1 and the reduction of binding by Nfia gene silencing.6?Silencing of Sfrp1 gene can inhibit the differentiation of adipocytes;Further studies have shown that the effect of NFIA on the differentiation of adipocyte is blocked in the context of Sfrp1 gene silencing.Conclusion: 1 ? The mRNA of Nfia expressed in adipose tissue was highly expressed in tissues of 4-week-old C57 BL / 6J mice,suggesting that NFIA might be involved in regulating adipogenesis.2?NFIA promotes the differentiation of precursor cells into adipocytes.3?NFIA transcriptionally activates Sfrp1 expression and inhibits the Wnt/ ?-catenin signaling pathway,thus promoting precursor cell adipogenic differentiation.