Experimental Study On The Regulation Of Osteogenic Differentiation By Mechanoresponsive Gene ATP13a3

Objective: The aim of this study was to verify the expression difference of ATP13a3 in the MC3T3-E1 cells under mechanical load,and then to analyze the effect of ATP13a3 on osteogenic differentiation of MC3T3-E1 cells under mechanical load,and explore the mechanism of the mechanoresponsive gene ATP13a3 in regulating osteogenic differentiation.Methods: 1.The expression of ATP13a3 in MC3T3-E1 cells was detected by q PCR under mechanical load(2500??,0.5Hz for 8h)and non mechanical load.2.Construction of the ATP13a3 gene overexpression vector and screening of the best siRNA for ATP13a3.3.Transfection of MC3T3-E1 cells with siRNA and overexpression vector were used to down-regulate or up-regulate the expression of ATP13a3,and mechanical load stimulation was given for 48 hours after transfection.4.Exploring the effect of ATP13a3 on osteogenic differentiation by detecting the activity of alkaline phosphatase,the expression of osteogenic differentiation marker genes and osteogenic differentiation marker proteins.5.In order to study the mechanism of ATP13a3 regulating osteogenic differentiation,transcriptome sequencing was performed on the mechanical load group and the mechanical load transfected ATP13a3 siRNA group.Bioinformatics and q PCR determined the mechanism of ATP13a3 in the process of mechanical load regulating osteogenic differentiation.Results: 1.The expression of ATP13a3 mRNA in MC3T3-E1 cells was down regulated under mechanical load.2.When MC3T3-E1 cells were given mechanical load and ATP13a3 down-regulation,the supernatant alkaline phosphatase activity of MC3T3-E1 cells was up-regulated,the expression of osteogenic differentiation genes Col1,OCN,and ALP increased,the expression of osteogenic differentiation proteins Col1,BMP2,and Runx2 increased.When MC3T3-E1 cells were given mechanical load and ATP13a3 up-regulation,there was no difference in the activity of alkaline phosphatase in the supernatant of MC3T3-E1 cells,The expression of osteogenic differentiation genes Col1,OCN,ALP and the expression of osteogenic differentiation proteins Col1,BMP2 and Runx2 had no significant change.3.Transcriptome sequencing was performed on the mechanical load group and the mechanical load transfected ATP13a3 siRNA group.Bioinformatics analysis shows that ATP13a3 promoted osteogenic differentiation mainly through TNF signaling pathway,and the key downstream gene might be Il6.4.Compared with MC3T3-E1 cell mechanical loading group,the ATP13a3 downstream genes Tnf,Tnfr2,c-Jun,Nfkb1,and Traf1 were up-regulated in MC3T3-E1 cell mechanical loading group and ATP13a3 siRNA group.Conclusion: ATP13a3 gene was a mechanoresponsive gene.Mechanoresponsive gene ATP13a3 could promote the osteogenic differentiation of MC3T3-E1 cells when it is down expressed.Under mechanical loading,low expression of ATP13a3 promoted osteogenic differentiation mainly by activating TNF signaling pathway.The downstream target genes of ATP13a3 may be Tnf,Tnfr2,c-jun,Nfkb1 and Traf1.