| In this study, Agilent SurePrint G3Mouse GE8*60K Microarray was employed toexplore J1mouse embryonic stem cells（J1mESCs）pluripotency genes after treated withCHIR99021for24hours. Thirteen differnetially expressed genes were validated by real timequantitative PCR (RT-qPCR). Systematic cluster analysis was performed using Cluster3.0andTreeView software. Gene Ontology (GO) annotation was performed using DAVID Database.The results of our study are following:1. A total of812(p<0.05, foldchange>2) and98(p<0.01, foldchange>5) transcripts weredifferentially expressed in CHIR99021treated J1mESCs compared with control. Theup-regulation of326genes and the down-regulation of486out of the812investigated geneswere found in differentially expressed gene. The up-regulation of55genes and thedown-regulation of43out of the98investigated genes were found in differentially expressedgene. RT-qPCR confirmed that gene expression styles are almost of no difference.2. The systematic cluster analysis showed that microarray experiment data had goodrepeatability, and the differentially expressed genes could be divided into two obviouslygroups.3. GO analysis showed that differentially expressed genes enriched in extracellularregion/matrix and basement membrance. These caused calcium binding, cofactor binding,sequence-specific DNA bingding, and transcription factor activity. The biological processanalysis showed that differentially espressed genes mainly caused neuron differentiation, cellgrowth, T cell activation during immune response. cellular amino acid biosynthetic process,cellular component size, and response to cAMP,biological adhesion,cell adhesion,developmental growth,embryonic organ developmen. Gene Functional Classification divideddifferentially expressed genes into two groups, one group including Pitx1、Hoxd9、Nkx1-2、Hoxc9、 Hoxd4.Another group including Gpr182、 Fzd10、 Cdh10、 Slamf9、 Ptger、24632428N05Rik、Efnb2、Cd96、Ms4a4d、Gpm6a.4. We cloned mouse Klf4promoter and analyzed the activity of klf4promoter by dual luciferase reporter assay. The results demonstrated that CHIR99021facilitated Klf4expression,which were confirmed by RT-qPCR, Western Blot, Immuno-fluorescence stainingand dual luciferase reporter assay.5. To explore the mechanism of Klf4expression regulation by CHIR99021, we firstproved that CHIR99021could obviously activate the classical Wnt pathway reporter system.According to the previous research about the consensus binding sites of classical Wntpathway and bioinformatics software prediction, we found two probable binding sites ofTCF/LEF in-1812/-1804and-1102/-1095region respectively. The dual luciferase reporterassay of the truncated Klf4promoter indicated that-1102/-1095region respond to CHIR99021stimulation.6. To further investigate that CHIR99021enhanced klf4expression by classical Wntpathway. We construced the eukaryotic expression vector of-catenin, which is the keyprotein of classical Wnt pathway. RT-qPCR and Western Blot showed that CHIR99021increased-catenin expression. Forced expression of-catenin in F9cells also activatedclassical Wnt pathway reporter vector and increased Klf4expression.Taken together, in this study we first demonstrated that CHIR99021promoted Klf4expression through classical Wnt pathway, and found a Wnt responsive element(-1102/-1095)on Klf4promoter. AP stain indicated that CHIR99021inhibited mouse embryonic stem cellsdifferentiation induced by Retinoic Acid. These results provided new insights intopluripotency mantainence of stem cells and induced pluripotency stem cells. |
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