Regulation Of Lineage Commitment Of Marrow Stromal Progenitor Cells By The Transcription Factor NFIX And The Mechanism Involved

Objective: NFIX is one of nuclear factor I family,which contains NFIA,NFIB,NFIC three others.NFI proteins could activate or inhibit the transcription of target genes by binding to the promoter of target genes.NFI play critical roles in many biological processes.A research had found that,growth delay and severe bone dysplasia were found in Nfix knockout mice,mainly manifested as cartilage endoossification damage,mineralization delay,spinal deformation,vertebral trabecular bone and cortical bone thinning,epiphyseal growth plate enlargement,trabecular bone mass reduction,and bone formation damage.This suggested that NFIX may be involved in bone development regulation.However,whether NIFX could regulate osteogenic differentiation of bone marrow stromal stem cells has not been reported.Therefore,our work mainly investigated the effect of NFIX on osteogenesis or adipogenesis,and figure out the potential mechanisms.Methods: 1.q RT-PCR was used to detect the m RNA level of NFIX in the tissues of C57BL/6J mice,the bone tissues of the elderly mice and OVX mice,as well as during the process of adipogenic and osteogenic differentiation of ST2 cells.2.Oil red O staining,q RT-PCR and WB were used to investigate the effect of NFIX on adipogenesis after overexpression or inhibition of NFIX in ST2 or C3H10T1/2.3.ALP staining,q RT-PCR and WB were used to investigate the effects of NFIX on osteogenesis after overexpression or inhibition of NFIX in ST2 or MC3T3-E1.4.Nfix sh RNA lentiviral plasmid were constructed,and the lentiviruses were packaged in 293 T cells.BMSCs was infected by Nfix-sh RNA LV,followed by adipogenic or osteogenic treatment.Oil red O staining,ALP staining,q RT-PCR and WB were used to investigate the effect of Nfix-sh RNA LV on adipogenesis and osteogenesis.5.RNA sequencing was used to screen the target genes of NFIX by Shanghai Meiji biological company,and results were verified by q RT-PCR.6.The promoter of Hmga1(high-mobility group A1,Hmga1)were analyzed by the TFbind database.The dual-luciferase reporter assay was used to detect the transtriptional activity of Nfix on Hmga1 promoter.7.Hmga1 overexpression plasmid was constructed.The effects of HMGA1 on osteogenesis and adipogenesis were determined by q RT-PCR,WB,oil red O staining/ALP staining.8.WB was used to detect the protein level of canonical Wnt/?-catenin pathway related genes after overexpression of HMGA1 and NFIX or inhibition of NFIX.Results: 1.NFIX was highly expressed in muscle,brain,heart,bone,perirenal fat,inguinal fat,brown fat and liver in C57BL/6J mice.The m RNA level of Nfix was decreased significantly in the old mice and the OVX mice.The m RNA level of Nfix was increased and reached the peak at the 7th day after osteogenic treatment.The m RNA level of Nfix was also increased and reached the peak at 3d after adipogenic treatment.2.The m RNA and protein level of Runx2,Ostrix,ALP,and OPN were significantly increased after overexpression of NFIX in ST2 and MC3T3-E1 cells;If NFIX was inhibited,the expression of these genes was significantly decreased.3.The expression of adipogenesis specific genes were significantly decreased after overexpression of NFIX in ST2 and C3H10T1/2 cells.However,the expression of them were significantly increased after NFIX was inhibited.4.Nfix-sh RNA LV could effectively inhibit the endogenous NFIX after infected BMSCs.It could damage the osteogenesis of BMSCs,but promote the adipogenesis.5.RNA-seq showed that there are 621 genes' expression altered,362 genes of them were upregulated including HMGA1.The m RNA of Hmga1 was upregulated after NFIX overexpression.6.Dualluciferase reporter assay demonstrated that NFIX could improve the transcription of Hmga1.7.The m RNA and protein level of osteogenesis specific transcription factors were significantly increased,the m RNA and protein level of adipogenesis specific genes were significantly decreased after overexpression of HMGA1.8.The protein level of p-LRP6,p-GSK3?(Ser9),TCF7L2,non-p-?-catenin were upregulated significantly after overexpression of HMGA1 or NFIX,while the protein level of tLRP6,t-GSK3?(Ser9)and t-?-catenin did not change a lot.The expression of these genes were downregulated after inhibition of NFIX,while the protein level of t-LRP6,t-GSK3?(Ser9)and t-?-catenin did not change significantly.Conclusion:1.The m RNA of NFIX was highly expressed in bone and adipose tissues of C57BL/6J mice at the age of 6 weeks,and the m RNA of NFIX was reduced in the bone of old mice and OVX mice,suggesting that NFIX may be involved in the regulation of osteoblast and adipocyte differentiation.2.NFIX could promote the osteogenesis of bone marrow stromal stem cells but inhibit the adipogenesis.3.NFIX could activate the transcription of HMGA1,in turn activate the canonical Wnt/?-catenin signaling pathway to promote the differentiation of bone marrow stromal stem cells into osteoblasts and inhibit the differentiation into adipocytes.