Mechanism Of ARHGAP44 Gene Regulating P53/C-myc/Cyclin D1 Pathway In The Proliferation,invasion And Migration Of Osteosarcoma

ObjectiveThis study investigates the expression of ARHGAP44 gene in osteosarcoma and how it affects the proliferation,invasion and migration of osteosarcoma cells by regulating the p53/C-myc/Cyclin D1 signalling pathway through various biological techniques at the clinical specimen,cellular and protein expression levels,providing a new therapeutic idea and theoretical basis for targeted treatment of osteosarcoma.MethodsIn this experiment,the expression level of ARHGAP44 gene in osteosarcoma was firstly detected using GEO database with immunohistochemical staining of clinical specimens,and its relationship with tumour prognosis was analysed.After constructing ARHGAP44 knockdown cell models(MG63 and HOS cell lines)and verifying the knockdown efficiency using q RT-PCR,we investigated the effects of ARHGAP44 gene on proliferation,invasion and migration of osteosarcoma cells through CCK-8 assay,clone formation assay,Transwell invasion assay and Wound healing scratch assay.The effect of ARHGAP44 on the proliferation,invasion and migration of osteosarcoma cells was investigated using Western Blot.In addition,the protein expression of ARHGAP44 gene and p53,C-myc and Cyclin D1 in osteosarcoma was examined by Western Blot technique to further clarify the logical relationship between ARHGAP44 gene and p53/C-myc/Cyclin D1 pathway.ResultsThe results of the GEO database data showed that the ARHGAP44 gene was highly expressed in osteosarcoma and was associated with poor prognosis in osteosarcoma patients(p=0.011)and negatively correlated with the expression of p53(p<0.001).Immunohistochemical staining showed that ARHGAP44 gene expression was significantly higher in tumour tissue compared to paraneoplastic tissue from osteosarcoma.CCK-8 and clone formation assays showed that downregulation of ARHGAP44 expression significantly reduced the proliferative capacity of MG63 and HOS cell lines(p<0.01).The results of Transwell invasion assay suggested that the invasion ability of MG63 with HOS osteosarcoma cell line was significantly reduced after down-regulation of ARHGAP44 gene expression(p<0.01).the results of Wound healing scratch assay showed that the migration ability of MG63 with HOS osteosarcoma cell line was significantly reduced after down-regulation of ARHGAP44 gene(p<0.01).The results of Western Blot illustrated that when the expression of ARHGAP44 gene was down-regulated,the expression levels of p53 in MG63 and HOS osteosarcoma cell lines were significantly higher compared to the control group,while the expression of C-myc and Cyclin D1 were significantly lower(p<0.05).This further suggests that down-regulation of ARHGAP44 may inhibit the proliferation,invasion and migration of osteosarcoma cells by regulating the p53/C-myc/Cyclin D1 signaling pathway.ConclusionsIn this study,we found that ARHGAP44 gene was highly expressed in osteosarcoma and was associated with poor prognosis of osteosarcoma.Downregulation of ARHGAP44 expression reduced the proliferation,invasion and migration of osteosarcoma.The results suggest that downregulation of ARHGAP44 may inhibit the malignant progression of osteosarcoma by regulating the p53/C-myc/Cyclin D1 signaling pathway,and ARHGAP44 may be a potential target for targeted therapy in osteosarcoma.