The Role Of Histone Demethylase KDM7A In Adipogenic And Osteogenic Commitment Of Mesenchymal Stem Cells And The Mechanisms Invloved

Backgrounds: Bone marrow mesenchymal stem/stromal cells(MSCs)are multipotent progenitor cells with multilineage differentiation potentials including osteogenesis and adipogenesis.Normal bone homeostasis relies on the inverse relation between adipogenesis and osteogenesis in MSCs.MSCs represent a reliable resource for tissue regeneration and periodontitis.While significant progress has been made in understanding transcriptional controls of MSC fate,little is known about how MSCs differentiation is epigenetically regulated.Since histone demethylases are the important part of Epigenetics,KDM7 A may present significant roles during the differentiation of MSCs.Methods:1.The expression levels of Kdm7 a were examined in different tissues.Various tissues were collected from 6-week old C57BL/6J mice and q RT-PCR was used to detect the m RNA expression of Kdm7 a in different tissues.2.The m RNA expression of Kdm7 a during adipogenic and osteogenic differentiation.q RT-PCR was used to detect the m RNA expression of Kdm7 a during adipogenic and osteogenic differentiation of ST2 cells.q RT-PCR was used to detect the m RNA expression of Kdm7 a during adipogenic differentiation of primary marrow stromal cells.3.A catalytically-dead Kdma7 a mutant plasmid was constructed.ST2 cells were transfected with vector,Kdm7 a wild type plasmid and catalytically-dead Kdm7 a mutant plasmid.Adipogenic or osteogenic induction was performed to allow the cells to differentiate.Oil-red O staining and ALP staining were used to observe differentiation.Then m RNA and protein were extracted.q RT-PCR and western blot were used to determine the gene and protein expression of adipogenic and osteogenic markers.4.Kdm7 a si RNA was designed.ST2 cells were transfected with negative control,Kdm7 a si RNAs.Oil-red O staining,ALP,q RT-PCR and western blot were used to determine the role of Kdm7 a si RNA on the differentiation.5.A Kdm7 a knock-down lentiviral plasmid was constructed.The lentiviruses were packaged in 293 T cells.ST2 cells were transfected with Kdm7 a knock-down lentiviruses.Oil-red O staining,ALP,q RT-PCR and western blot were used to determine the role of lentiviruses on the differentiation.6.Primary marrow stromal cells were transfected with Kdm7 a knock-down lentiviruses.Adipogenic or osteogenic induction was performed to allow the cells to differentiate.Oil-red O staining,ALP and q RT-PCR were used to determine the role of lentiviruses on the differentiation.7.The endogenous expression of Kdm7 a was inhibited by si RNA in ST2 cells.Without induction,q RT-PCR was used to determine the m RNA expression of adipogenic and osteogenic markers,C/EBP??C/EBP??PPAR?,Sfrp1?Klf7?Klf9.8.The endogenous expression of Kdm7 a was inhibited by si RNA in ST2 cells.Without induction,chromatin immunoprecipitation(Ch IP)method was used to determine if KDM7 A can bind to the TSS region of C/EBP? and Sfrp1,and the change of binding of histone H3K9me2,H3K27me2 on C/EBP? and Sfrp1 chromatin.Results:1.Kdm7 a m RNA was highly expressed in bone,and relatively low in intestines.2.q RT-PCR analysis demonstrated that Kdm7 a expression was incresaed in primary marrow stromal cells and ST2 cells after adipogenic treatment,also incresaed in ST2 cells after osteogenic treatment.3.Silencing of endogenous Kdm7 a by si RNA in ST2 cells inhibited adipogenic differentiation.Compared with negative control,Kdm7 a si RNA led to a significant decrease in the number of differentiated adipocytes.Accordingly,the m RNA and protein levels of adipogenic transcription factors and marker genes were decreased.4.Silencing of endogenous Kdm7 a by si RNA in ST2 cells induced osteogenic differentiation.Compared with negative control,Kdm7 a si RNA led to a significant increase in the number of differentiated osteogenesis.Accordingly,the m RNA and protein levels of osteogenic transcription factors and marker genes were increased.5.Wide type Kdm7 a overexpression in ST2 cells induced adipogenic differentiation,and reduced osteogenic differentiation.While mutant Kdm7 a overexpression didn't work.6.Silencing of endogenous Kdm7 a by lentiviruses in ST2 cells and primary marrow stromal cells decrease adipogenic differentiation,but increase osteogenic differentiation.7.Silencing of endogenous Kdm7 a in ST2 cells,the m RNA expression of C/EBP? and Sfrp1 had a significant decrease without induction.8.Silencing of endogenous Kdm7 a in ST2 cells,Ch IP datas demonstrated that,the binding of KDM7 A on the TSS region of C/EBP? and Sfrp1 was reduced,but the binding of H3K9me2 and H3K27me2 were increased.Conclusions:1.The expression levels of Kdm7 a in different tissues were various.2.Kdm7 a may have a regulatory role in adipogenesis and osteogenesis in primary marrow stromal cells and ST2 cells.3.In vitro,Kdm7 a may induce adipogenic differentiation,and inhibit osteogenic differentiation in MSCs.4.KDM7 A may function dependently from its demethylase activity.5.KDM7 A may control C/EBP? and Sfrp1 expression by removing H3K9me2 and H3K27me2 on their promoters.