The Effect And Mechanism Of Knockdown ZNF503 On Osteogenic/adipogenic Differentiation Of HBMSC

Objective: To understand the effect of knockdown Zinc finger protein 503(ZNF503)on osteogenic/adipogenic differentiation of human bone marrow mesenchymal stem cell(h BMSC),and verify whether knockdown ZNF503 can promote osteogenic differentiation and inhibit adipogenic differentiation of h BMSC.The aim of this study is to investigate preliminarily and verify the molecular mechanism of ZNF503 on h BMSC differentiation direction.Methods: The sh ZNF503 lentivirus vector was constructed by p LKO-Tet-On,and h BMSC infected with the disease venom was collected.The cells statically expressing ZNF503-sh RNA were screened out,and the knockdown efficiency of ZNF503 was detected by q RT-PCR.Osteogenic/adipogenic differentiation were induced in both sh NC and sh ZNF503 group,and the formation of intracellular bone nodules and lipid droplets was observed by alizarin red staining and oil-red O staining.The m RNA expressions of Runx2,PPAR-γ,CEBP-α and ZNF503 were detected by q RT-PCR.The protein expressions of ZNF503 were detected by WB.RNA of h BMSC in sh NC and sh ZNF503 groups at 0 and 16 days of osteogenic/adipogenic differentiation was extracted for high-throughput sequencing to find differentially expressed genes.GO enrichment analysis and KEGG pathway analysis were performed,and the expression levels of major differentially expressed genes were detected by q RT-PCR.Results:(1)Compared with sh NC group,the knockdown efficiency of sh ZNF503 was 35.6%.On day 16 of osteogenic differentiation,bone nodule formation was significantly increased in sh ZNF503 group compared with sh NC group.The m RNA expression of Runx2 in sh ZNF503 group on 16 d were higher than those in sh NC group.On both day 0 and 16 of osteogenic differentiation,the m RNA and protein level of ZNF503 in sh ZNF503 group were lower than those in sh NC group.On day 16 of adipogenic differentiation,the lipid droplets aggregation in ZNF503 group was significantly increased compared with sh NC group.On day 16 of adipogenic differentiation,PPAR-γ and CEBP-α m RNA was higher in both sh NC and sh ZNF503 group compared with 0 d.At 0 and 16 days of adipogenic induction,the m RNA and protein expression of ZNF503 in sh ZNF503 group decreased compared with the sh NC group.(2)There were 901 differentially expressed genes in sh NC-16 d vs sh NC-0d during osteogenic differentiation of h BMSC,and 803 differentially expressed genes in sh ZNF503-16 d vs sh ZNF503-0d.Differential genes are significantly enriched in many important processes such as cell proliferation,extracellular matrix and extracellular matrix structural constituent during osteogenic differentiation,and enriched in complement and coagulation cascades,transcriptional misregulation in cancer,Fox O signaling pathway and other signaling pathways.There were 829 differentially expressed genes in sh NC-16 d vs sh NC-0d during adipogenic differentiation of h BMSC,and 729 differentially expressed genes in sh ZNF503-16 d vs sh ZNF503-0d.Differential genes are significantly enriched in tissue development,collagen-containing extracellular matrix,glycosaminoglycan binding and other biological process,and enriched in PPAR signaling pathway,Rap1 signaling pathway and drug metabolism-cytochrome P450 and other signaling pathways.(3)On 16 d of osteogenesis differentiation,the m RNA and FKPM levels of sh ZNF503 group were lower than those of sh NC group,and MGP m RNA and FKPM levels of sh ZNF503 group were higher than those in 0 d.But the m RNA and FKPM levels of ITGA2 and SEMA3 A in sh NC and sh ZNF503 group were also lower than 0 d after osteogenic differentiation.On 16 d of adipogenic differentiation,the m RNA and FKPM levels of h BMSC in sh ZNF503 group were lower than sh NC group,and the m RNA and FKPM levels of TIAM1 and ADAMTS6 in sh NC and sh ZNF503 group were lower than those in 0 d.However,the m RNA and FKPM levels of LBP in sh NC and sh ZNF503 group were lower than sh NC group on 16 d after adipogenic differentiation.Conclusion:(1)Knockdown of ZNF503 can promote osteogenic differentiation and inhibit adipogenic differentiation of h BMSC.(2)Knockdown of ZNF503 may promote osteogenic differentiation of h BMSC by up-regulating the expression of MGP,SEMA3 A,ITGA2 and other genes.(3)Knockdown of ZNF503 may inhibit adipogenic differentiation of h BMSC by up-regulating the expression of TIAM1,ADAMTS6 and down-regulating the expression of LBP and other genes.