The Role And Mechanism Of Tensile Force On Osteogenic Differentiation Of Valvular Interstitial Cells

Objective: To explore the effect of tensile force on osteogenic differentiation of aortic valve interstitial cells(VICs)and its mechanism.Methods: Samples of porcine aortic valves were obtained,and primary porcine VICs(pVICs)were isolated and identified by immunofluorescence.pVICs were stimulated under stretch of different amplitude and time.The expression of osteogenic differentiation markers was detected by RT-q PCR and western blot.Samples of human aortic valves were obtained,and primary human VICs(hVICs)were isolated and identified by immunofluorescence.hVICs were stimulated under 10%-60 min stretch,and the control group were cultivated without stretch.The expression of osteogenic differentiation markers was detected by western blot.Alizarin red staining was used for histological staining of calcium deposition.The differential genes between two groups were detected by m RNA-seq,and the possible key genes were screened by KEGG and GO enrichment analysis.The genes was verified by RT-q PCR and the differential gene TUBB3 was selected.The effect of tensile force on the expression of TUBB3 was detected after the cells were stimulated under stretch of different amplitude and time.The effects of RNA silencing of TUBB3 on osteogenic differentiation markers and calcium deposition under stretch were detected by western blot and alizarin red staining.RT-q PCR was used to detect the gene expression of histone acetyltransferases and deacetylases of hVICs under10%-60 min stretch.Western blot was used to detect the level of H3K9 ac under stretch of different amplitude and time.MG149 was used to inhibit the level of H3K9 ac.Then western blot was used to detect the effects of MG149 on osteogenic differentiation markers,TUBB3 and H3K9 ac under stretch,and alizarin red staining was used to detect calcium deposition.Finally,the effects of stretch and MG149 on the level of H3K9 ac in the promoter region of the TUBB3 were detected by Ch IP-q PCR.Results: 10%-60 min stretch can promote the expression of osteogenic differentiation markers in pVICs.10%-60 min stretch can promote the expression of osteogenic differentiation markers and TUBB3,and can aggravate calcium deposition in hVICs.The expression of TUBB3 is different under stretch of different amplitude and time.Silencing of TUBB3 via si RNA can inhibit the expression of osteogenic differentiation markers and calcium deposition promoted by tensile force.10%-60 min stretch can increase the level of H3K9 ac in hVICs,and the level of H3K9 ac is different under stretch of different amplitude and time.MG149 can inhibit the expression of osteogenic differentiation markers and calcium deposition of hVICs under stretch.10%-60 min stretch can increase the level of H3K9 ac in TUBB3 promoter region and promote its transcription,while MG149 has the opposite effect.Conclusion: 10%-60 min stretch can promote the osteogenic differentiation of pVICs.10%-60 min stretch can promote TUBB3 expression by increasing the level of H3K9 ac in the TUBB3 promoter region,and promote osteogenic differentiation and calcium deposition of hVICs through TUBB3,while MG149 has the opposite effect.