| Objective:Adipose-derived stem cells(ADSCs)have the potential of multi-directional differentiation.And it is the key to achieving adipose tissue reconstruction by inducing ADSCs differentiating into adipocytes,which is of great significance to repairing huge wounds and breast defects.However,the adipogenesis of ADSCs has not been clearly understood,and there are problems such as low conversion efficiency and low survival rate of regenerated adipose tissue in practical application.Therefore,based on the gene chip data of adipogenesis of ADSCs,this study explored the genes related to adipogenic differentiation of ADSCs through bioinformatics analysis and further validated their functions with functional study to promote clinical transformation of adipose-tissue engineering.Methods:This study was mainly divided into two parts.In the first part,we mainly analyzed three Gene chip data(GSE37836,GSE41352,and GSE44303)representing different stages of the adipogeneic process of ADSCs,and we transformed the three Gene chip data into gene symbols.The gene co-expression network was constructed with Cytoscape software.Differential genes expression analysis was performed(FDR<0.05 or fold change>2)and a protein-protein interaction network was constructed.Further,weighted gene co-expression analysis,function enrichment analysis and KEGG pathway enrichment analysis were performed.In the second part,we designed and verified the best small interfering RNA(siRNA)sequences of JUN and FOS.Then we knocked down the JUN and FOS genes by the best siRNA,and detected the lipid droplet formation,the expression level of genes related to PPARy signaling pathway,inflammation and apoptosis levels of ADSCs during the adipogeneic process.Simultaneously,we established an inflammation model by stimulating ADSCs with LPS at a concentration of 20 ng/μl,so as to further detect the effect of JUN and FOS on the inflammatory response and apoptosis of ADSCs.Results:There were about 500 differentially expressed genes in GSE37836(311 up-regulated and 189 down-regulated),489 differentially expressed genes in GSE41352(247 up-regulated,242 down-regulated),and 261 differentially expressed genes in GSE44303(183 up-regulated,78 down-regulated).The results of functional enrichment analysis suggested that the differentially expressed genes were mainly involved in lipid metabolism,triglycerides and lipid synthesis and decomposition and cellular metabolic responses.The KEGG signal pathway enrichment analysis results showed that the differentially expressed genes were mainly related to tyrosine metabolism,PPARy signal pathway,and oxidative stress-related signal pathways in cells.The protein-protein interaction network constructed by the STRING tool showed that the mainly concentrated codes were VCAM1,JUNB,FOS,NR4A1,SGK1.The weighted gene co-expression analysis results showed the co-expressed genes of brown module in GSE37836,the magenta module and the brown module in GSE41352,and the turquoise module in GSE44303 were significantly related to the adipogenesis of ADSCs,which were involved in cell metabolism,energy conversion,apoptosis and inflammation regulation.It was important that AP-1(homologous or heterodimer of JUN and FOS)was included in the above-mentioned significant modules.For the ADSCs transfected with FOS siRNAl(GGAACAGUUAUCUCCAGAA)and JUN siRNAl(UCUACGCAAACCUCAGCAA),both the mRNA transcription level and protein expression level of FOS and JUN were the lowest,so they were chosen as the best interference sequences.After using the above sequences to interfere with JUN and FOS respectively,oil red O staining results showed that lipid droplet formation was reduced during the adipogeneic process,and the mRNA transcription level and protein expression level of the important transcription factors PPARy and the genes related to PPARy singling pathway(PPARGCa,FABP7,AQP7)were also significantly reduced.During the adipogeneic process,the mRNA transcription level of inflammatory factors IL-2,IL-6 and TNFa showed an increasing trend.After interfering with the expression and activity of AP-1,the mRNA transcription level of IL-2,IL-6 and TNFα increased compared with the control group.Meanwhile,The proliferated ability of ADSCs showed a decreasing trend,and the proliferation of ADSCs increased slightly compared with the control group after knockdown of the FOS and JUN gene and the addition of curcumin.On the 12th day of the adipogeneic process,the apoptosis level of ADSCs in FOS and JUN gene knockdown group and curcumin added group was significantly reduced.We successfully established an inflammation model by stimulating ADSCs with LPS at a concentration of 20 ng/μl for 6 hours,and the protein expressed level of inflammatory factors(IL-2,IL-6,and TNFα)were significantly increased.Compared with the control group,the protein expressed level of IL-2,IL-6,and TNFa in the JUN and FOS knockdown group and the curcumin-added group were significantly increased.In addition,the results of TUNEL staining and Annexin V-FITC flow cytometry showed that the apoptosis rate of ADSCs was reduced after inhibiting the expression and activity of AP-1.What’s more,the mRNA transcription and protein expression level of apoptosis factors Caspase3,Caspase9 and Bax were significantly reduced,but the anti-apoptotic factor Bcl-2 was increased.Conclusions:The results of the bioinformatics analysis indicated that AP-1 was significantly related to the adipogenesis of ADSCs,and the PPARy signaling pathway may be one of the key pathways for this biological process.At the same time,AP-1 may be involved in regulation of the apoptosis level and inflammatory response of ADSCs during adipogenesis.Inhibiting the expression and activity of AP-1 could inhibit the adipogenesis of ADSCs,which was involved with the PPARy signaling pathway.After Inhibiting the expression and activity of AP-1,the expressed level of inflammatory factors was increased,but the level of apoptosis was decreased accompanied by the decreased expressed level of apoptotic factors and the increased expressed level of anti-apoptotic factors respectively. |
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