Effect Of Piezo1 Mechanically Sensitive Ion Channel Mediated Fluid Shear Force On Migration In MC3T3-E1 Osteoblasts

Objective:To discuss the variations of the expression of piezo1 mechanically sensitive ion protein in MC3T3-E1 osteoblasts with the increasing loading time of fluid shear stress(FSS)and the effect of Piezo1 protein in migration process of mouse MC3T3-E1 osteoblast cells.Methods:With the use of a self-designed parallel plate flow system,FSS(12dyn/cm~2)was acted on MC3T3-E1 osteoblasts for 0,15,30,45,60,90 min.The expression level of piezo1 protein was detected by immunofluorescence and Western blot.Results Piezo1 was obviously expressed in the cytoplasm and nucleus of osteoblasts,especially in the cytoplasm.FSS of 12 dyn/cm~2 was acted on MC3T3-E1cells cultured in vitro.With the increase of loading time,the expression of piezo1protein was upregulated.At 45 minutes after loading,the expression of piezo1 protein reached the maximum.The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA(siRNA)transfection group(group A),negative control group(group B),and blank control group(group C).Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent,respectively;group C was only added with siRNA transfection reagent;and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope,and the transfection efficiency was calculated.The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot.Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection.Results:Loading 12 dyn/cm~2 FSS can upgrade the expression of piezo1mechanosensitive ion protein in MC3T3-E1 osteoblasts,and the optimal loading time of FSS is 45 minutes.After 48 hours of transfection,group A showed a slight increase in cell volume and mutant growth,but cell colonies decreased,suspension cells increased and cell fragments increased when compared with untransfected cells.Under fluorescence microscope,green fluorescence was observed in MC3T3-E1osteoblasts of group B,and the transfection efficiency was 68.56%±4.12%.Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C(P<0.05);there was no significant difference between group B and group C(P>0.05).Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C(P<0.05);there was no significant difference between group B and group C(P>0.05).Conclusion:Loading 12 dyn/cm2 FSS can upgrade the expression of piezo1mechanosensitive ion protein in MC3T3-E1 osteoblasts,and the optimal loading time of FSS is 45 minutes.Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.