KLF2 Transcription Factor Promotes The Stemness Of Human Bone Marrow Mesenchymal Stem Cells By Regulating FGFR3 Gene

Objective:In recent years,human bone marrow mesenchymal stem cells(h MSCs)have been widely concerned.KLF2 is a transcription factor related to the proliferation and differentiation of embryonic stem cells(h ESCs).It has been reported that KLF2 plays an important role in maintaining the ability of self-renewal and proliferation and differentiation of ESCs.It has also been reported that KLF2 participates in the process of self-renewal and proliferation in mesenchymal stem cells(MSCs),but the mechanism of KLF2 in h MSCs is not clear.This study explored the role of KLF2 in stemness maintenance of h MSCs and the molecular mechanism of its regulation with related gene FGFR3,providing guidance for the transformation of MSCs in tissue engineering and clinical application.Method:Bone marrow mesenchymal stem cells were extracted from patients who voluntarily donated bone marrow in the First Affiliated Hospital of Zhejiang University Medical College from December 2014 to December 2019.After primary culture of BMSCs,FGFR3 gene was knocked out by interverning RNA,and then the knock-out efficiency was verified by RT-PCR.The related stemness genes(Nanog,Oct4,Sox2,rex1)and differentiation related genes(ALP,Sox9,PPAR-?2)were detected by RT-PCR to prove whether they have relevance.The proliferative activity and ability of the cells were tested by the cell clone formation experiment.The direct regulation of KLF2 transcription factor in the promoter of FGFR3 gene was also detected by chromatin immunoprecipitation assay(chip).Result:In previous studies,we found that when we knocked out KLF2 transcription factor with intervering RNA,the expression of target gene FGFR3 also decreased significantly,with convergence;then we knocked out the target gene FGFR3,and verified its knock-out efficiency by RT-PCR,the results showed that the expression of FGFR3 decreased significantly;after knocking out the FGFR3 gene,the stemness related genes(Nanog,Oct4,Sox2 The expression of rex1)was significantly lower than that of mock group,only ALP and Sox9 were up-regulated in differentiation related gene.The results of cell clone formation test showed that the cell proliferation ability of FGFR3 knockout group was significantly lower than that of mock group;from chip-sequence results,we can find that KLF2 was more likely to combine with FGFR3 promoter fragments of the second group and the fourth group,and both of them had relatively high binding rate.In some cases,KLF2 transcription factor would directly bind with FGFR3 to affect the stemness and proliferation of stem cells.Conclusions:KLF2 transcription factor can regulate the expression of FGFR3 gene to promote the stemness and proliferation of human bone marrow mesenchymal stem cells...