In Vitro Study On The Role Of Foxc2 In The Osteogenic Differentiation Of MC3T3-E1 Cells

Oral and maxillofacial bone defects caused by tumor,trauma and infection not only impair patients'chew,swallow and language functions,but also lead to facial deformities,all of above greatly reduce patients'life qualities and have serious impact on mental health.How to repair defects,how to promote new bone reconstruction and restore the oral-facial function and appearence in a better way,are always the problems that surgeons try to solute.Bone formation and remodeling is a special and complex process,osteoblast,which is the primary unit,plays an important role.It is well known that differentiation and proliferation of osteoblasts is mainly regulated by the transcription factors.Explore and study osteogenesis related transcription factors,reveal and perfect the underlined mechanism,which is benificial for providing some basis for the establishment of new treatment strategies.The forkhead box C2?Foxc2?protein is one member of the forkhead/winged helix transcription factor family,which plays an important role in regulation of embryonic development,angiogenesis,adipose and lymphoid tissue generation.However,The exact role of Foxc2 in the process of cell proliferation and osteogenetic differentiation requires further investigation.Objective 1.To observe the Foxc2 expression pattern under the stimulation of osteogenesis related factors?osteoblast inducing media,parathyroid hormone,simvastatin?.2.To observe the role of Foxc2 on cell proliferation,cell cycle,cell apoptosis and differential ablities in MC3T3-E1 cells and C3H10T1/2 cells.3.To screen and preliminarily verify candidate target genes which may subjected to Foxc2 regulation in cell proliferation and osteogenic differentiation.Methods 1.Observe Foxc2 expression pattern in MC3T3-E1 cells under the stimulation of osteogenesis related factor?osteoblast inducing media,parathyroid hormone,simvastatin?by Real-Time PCR and Western blotting.2.To observe the cell proliferation,cell cycle,cell apoptosis and differential ablities of MC3T3-E1^Foxc2+cells and C3H10T1/2^Foxc2+cells transfected with lentivirus vectors containing Foxc2 by CCK-8,FCM,Real-Time PCR and Western blotting,ALP/ARS staining,using MC3T3-E1^EGFP and C3H10T1/2^EGFP as control.Use siRNA to construct MC3T3-E1^si-Foxc2 as a reverse validation.3.Evaluate the global gene expression profiles of MC3T3-E1^Foxc2+and MC3T3-E1^EGFPGFP by genechip and bioinformatics analysis,and preliminary verify the expression level of genes of interested by Real-Time PCR.Results 1.Osteoblast inducing media,parathyroid hormone,and simvastatin can successfully induce osteogenic differentiation of MC3T3-E1 cells in vitro and significantly increase Foxc2 gene expression level in the cells.Foxc2 gene expression shows a characteristic of first rise after the fall during osteogenic differentiation.Strong osteogenesis stimulating factor-parathyroid hormone can make the expression of Foxc2 ahead of time,simvastatin stimulates Foxc2 expression in a pattern of concentration dependence.2.After Foxc2 overexpression lentivirus sucessfully transfect MC3T3-E1 cells and C3H10T1/2 cells,two kinds of cell proliferation activity have been significantly suppressed,a key osteogenic differentiation transcription factor-Runx2 and alkaline phosphatase expression levels increase obviously,prompting Foxc2 can effectively promote cells osteogenesis differentiate in vitro.On the other hand,Foxc2 expression significantly inhibited the key transcription factor Ppar-? expression and adipogenic differentiation of mouse embryonic stem cell lines C3H10T1/2 in vitro.3.Using Agilent gene chip detection,bioinformatics analysis and Real-Time PCR to compare the global gene expression profiles differences of MC3T3-E1^Foxc2+ and MC3T3-E1^EGFP,preliminary screening candidate target genes which may subjected to Foxc2 regulation in cell proliferation and osteogenic differentiation,such as Mmp9,Ereg,etc.Conclusions Osteoblast inducing media,parathyroid hormone,and simvastatin can successfully induce osteogenic differentiation of MC3T3-E1 cells in vitro and significantly increase Foxc2 gene expression level in the cells,which suggest Foxc2 is closely related to the osteogenic differentiation of preosteoblast.In this research,a series of molecular biology experiment after Foxc2 overexpression lentivirus transfected cells suggest that Foxc2 can effectively promote cells osteogenic differentiation in vitro by inhibition of cell proliferation,activation of Runx2 and ALP expression.Mmp family and Ereg may participate in Foxc2-regulates osteogenetic differentiation.