The Role Of LATS1 Gene Demethylation In Proliferation,Apoptosis And Invasion Of Human Osteosarcoma

To explore the role of LATS1 gene in osteosarcoma,and to increase the expression of LATS1 gene by demethylation drugs,so as to study its effect on the function of human osteosarcoma cells.Methods:(1)Immunohistochemical staining was used to detect the difference of LATS1 gene expression between human osteosarcoma and normal bone tissue.QRT-PCR was used to detect the expression of m RNA in human osteosarcoma tissue,normal bone tissue,osteoblast(Hfob1.19)and human osteosarcoma cell line(Saos2,U2).Western blotting(Western-Blot)was used to detect the difference of protein expression between osteoblasts(Hfob1.19)and human osteosarcoma cell lines(Saos2,U2).(2)Methylation specific PCR(Methylation-specific PCR,MSP)was used to detect the methylation status of LATS1 in human osteosarcoma cell lines(Saos2 and U2).Saos2 cells and U2 cells were treated with 5-Aza-2’Deoxycytidine.The control group,solvent group and experimental group were set up respectively.The m RNA expression of LATS1 in each group was detected by q RT-PCR,and the protein expression of LATS1 in each group was detected by Western-Blot.After drug treatment,the effects of LATS1 on the biological characteristics of osteosarcoma cells,such as proliferation(RTCA),apoptosis(flow cytometry)and invasion of(Transwell),were detected.Results:(1)The results of immunohistochemistry showed that LATS1 was expressed in cytoplasm,and the expression level in bone tissue was higher than that in osteosarcoma tissue(P < 0.0001).The results of q RT-PCR showed that the expression of LATS1 was low in osteosarcoma(P < 0.0001),and the expression of LATS1 was low in osteosarcoma cell lines Saos2 and U2(P < 0.001).The results of Western-Blot showed that the expression of LATS1 was low in osteosarcoma cell lines Saos2 and U2(P <0.001).(2)LATS1 gene was methylated in osteosarcoma cell lines Saos2 and U2.The results of q RT-PCR showed that the expression of LATS1 was high in Aza-Saos2 group and Aza-U2 group(P < 0.0001).The results of Western-Blot showed that LATS1 was highly expressed in Aza-Saos2(P < 0.001)and in Aza-U2 in experimental group(P <0.01).The results of RTCA showed that the proliferation of Aza-Saos2 and Aza-U2 in the experimental group were significantly lower than those in osteosarcoma Saos2 and U2cells(P < 0.0001),and the apoptosis of Aza-Saos2 and Aza-U2 in the experimental group were significantly higher than those in osteosarcoma Saos2 and U2 cells(P < 0.0001).The results of Transwell showed that compared with osteosarcoma Saos2 and U2 cells,the invasive ability of Aza-Saos2 and Aza-U2 in the experimental group were significantly decreased(p < 0.0001).The results of cell scratch assay showed that compared with osteosarcoma Saos2 and U2 cells,the migration of Aza-Saos2 in the experimental group was significantly weaker than that of Saos2(P < 0.001),and the migration rate of Aza-U2 in the experimental group was significantly lower than that of U2(P<0.001).Conclusion:(1)LATS1 gene plays an important role in the occurrence and development of osteosarcoma.(2)the degree of methylation of LATS1 gene was higher in osteosarcoma cells.(3)after demethylation treatment,the m RNA and protein levels of LATS1 gene were significantly up-regulated.(4)LATS1 gene can inhibit the proliferation of Saos2 and U2 cells,induce apoptosis and prevent their invasion.