Expression Of Piezo 1 And Its Impact On The Biological Fuction Of Osteosarcoma Cells

OBJECTIVE To study the expression of Piezo1 in the mechanical stretch stress models of the osteosarcoma cells,and to investigate the effect of shRNA-Piezo1 down-regulating the expression of Piezo1 on proliferation and apoptosis of osteosarcoma cells.METHODS Construct the Piezo1-shRNA lentivirus vector to ransfect the logarithmic growth of osteosarcoma MG63 and U2 cell lines;The experiment was divided into blank control group(no lentiviral transfected cells),negative control group(unordered sequence plasmid lentiviral transfected cells)and ShPiezo1 group(shRNA Piezo1 interference sequence plasmid lentiviral transfected cells);The cell model of mechanical tensile stress in vitro was constructed by using Flexcell cell tension loading system(set 0h,2h,12 h,24h,48 h mechanical tension stress groups according to the pre-experiment);Western-blot and RT-qPCR were used to detect the expression of the Piezo1 in osteosarcoma cells;The immunofluorescence was used to locate the expression of the Piezo1 protein in osteosarcoma cells;CCK-8 experiment was used to detect the proliferation of the osteosarcoma cells;Annexin V-PI was used to detect the apoptosis of the osteosarcoma cells.RESULTS(1)The lentivirus vectors were successfully constructed and successfully infected the osteosarcoma MG63 and U2 cell lines.The results of RT-qPCR and Western-blot showed that there were no difference in the expression of the two osteosarcoma cell lines,and the MG63 cell line was used as the cells for this experiment.(2)The results of RT-qPCR showed that the expression of Piezo1 was different in osteosarcoma cells under mechanical stretch stress at different time,and the expression was highest under 24 h mechanical stretch stress(P<0.05).The expression of Piezo1 all decreased compared with the blank control group in ShPiezo1 group(P<0.05).(3)The results of Western-blot had showedthe expression of the Piezo1 protein in the osteosarcoma cells under mechanical stretch stress at different time were different,and the expression were the highest under the mechanical stretch stress of 24h(P<0.05).No significant expression of Piezo1 protein was observed in the ShPiezo1 group.(4)the results of the immunofluorescence showed : the expression of Piezo1 protein in the osteosarcoma cell membrane was observed in the blank group,the expression of Piezo1 protein of the ShPiezo1 group was significantly lower than that in the blank group(P<0.05).(5)CCK-8 assay had showed the mechanical stretch stress of the osteosarcoma cells at different time affect the cells Proliferation rate.After loading 24 h mechanical stretch stress,the proliferation rate of the osteosarcoma cells was the lowest(P<0.05).The proliferation rate of osteosarcoma cells in ShPiezo1 group was significantly higher than that in the blank control group after 12 h,24h,48 h mechanical stretch stress(P<0.05).(6)The osteosarcoma cells apoptosis by Hoechst staining and Annexin V-PIstaining had showed the mechanical tensile stress at different time affect the apoptosis rate of the osteosarcoma cells.The early apoptosis rate of the osteosarcoma cells was highest after loading 2h mechanical stretch stress(P<0.05).After loading 24 h mechanical stretch stress,the cells late apoptosis rate was the highest(P<0.05),and the total apoptosis rate was the highest(P<0.05).In the ShPiezo1 group,the late apoptosis and total apoptosis rate of the cells decreased after loading 12 h,24h and 48 h mechanical stretch stress(P<0.05).CONCLUSION In vitro,the mechanically sensitive ion channel Piezo1 protein can express in human osteosarcoma cells.Piezo1 can inhibit the proliferation of osteosarcoma cells and promote the apoptosis of osteosarcoma cells.