The Role And Molecular Mechanism Of Simvastatin Regulating YAP-Mediated SOX9 Transcription In Inhibiting Osteosarcoma Metastasis

Background:Osteosarcoma is of high incidence,malignancy and metastasis risk,and the latter is an important factor leading to poor efficacy and dis-satisfactory prognosis in patients.Statins are mainly used to reduce blood lipid.The repression in tumor proliferation and metastasis of statins had also been reported recently,however,only several articles were found in study of osteosarcoma.Moreover,the highly actived level of both YAP and SOX9 were demonstrated to be positive with the migration and invasion of osteosarcoma.Besides,SOX9 was found possibly to be the direct target gene of YAP in our previous bioinformatics analysis.Therefore,the aim of our research is to verify whether simvastatin can inhibit the metastasis of osteosarcoma by regulating the YAP-mediated SOX9 transcription,and if so,its specific molecular mechanism will be further explored and investigated,in order to enrich the research content of osteosarcoma metastasis.Methods:Part ?: Firstly,we evaluated the activity of HMGCR(the target of simvastatin)in osteosarcoma cells,as well as the alterations of migration and invasion after receiving the treatment of simvastatin.Then,we assessed the relationship between SOX9 and the migration and invasion ability by knocking-down SOX9.Moreover,whether SOX9 expression can be suppressed by simvastatin in osteosarcoma cells was also tested.Finally,we conducted a dual luciferase labeling assay to assess whether simvastatin had inhibition effect on SOX9 promoter activity in osteosarcoma cells.Part ?: Firstly,the SOX9 promoter region was analyzed to predict the possible binding regions of TEAD and SOX9.Then,we applied a Ch IP assay to further screen and tried to find the most likely binding sites among the above-predicted ones.Moreover,by testing the changes of SOX9 promoter activity after deleting the corresponding binding regions,we accurately verified the specific binding sites.Finally,the expression of SOX9 after YAP or TEAD knockdown was also assessed.Part ?: Firstly,we evaluated YAP function in osteosarcoma cells by assessing its phosphorylation level and cell localization,as well as the changes of migration and invasion after the knocking-down of YAP.Secondly,the effects of simvastatin on YAP phosphorylation and cell localization were evaluated.Finally,the changes in the binding ability of YAP and SOX9 promoter after simvastatin treatment were also assessed by Ch IP test.Part ?: Firstly,the expression of RhoA in osteosarcoma cells,and the changes of RhoA activity after simvastatin treatment were evaluated.Secondly,after overexpression of RhoA,the effects of simvastatin on YAP activity and SOX9 expression were tested.Then,the effect of simvastatin on the cytoskeleton assembly was detected.Finally,YAP activity and SOX9 expression were detected after in the osteosarcoma cells were treated with RhoA inhibitor(C3 transferase)and cytoskeleton inhibitor(cytochalasin D).Part ?: By constructing the osteosarcoma-induced lung metastatic model of caudal vein in nude mice,we evaluate whether simvastatin affects the lung metastasis risk and the survival time of nude mice.Moreover,we used immunohistochemistry assay to assess the expression of RhoA,YAP and SOX9 in lung metastatic nodules.Results:Part ?: HMGCR was highly expressed in osteosarcoma cells,and simvastatin treatment showed a inhibitory effect on osteosarcoma cells' migration and invasion via down-regulating the mevalonate pathway.SOX9 was highly up-regulated and mediated a positive effect on osteosarcoma cells' migration,invasion and EMT process.By repressing mevalonate pathway,the intervention of simvastatin showed a negatively regulation on the transcriptional activity of SOX9.Part ?: SOX9 was the direct target gene of YAP-TEAD.The TB1 site in the SOX9 promoter region was necessary for TEAD to bind to SOX9 and regulate SOX9 transcriptional activity.Part ?: YAP is highly expressed in osteosarcoma cells,and showed a positive effect on osteosarcoma cells' migration,invasion and EMT process.By decreasing YAP activity via down-regulating mevalonate pathway,the migration and invasion were obviously suppressed by simvastatin treatment.Simvastatin regulated the activity of YAP to control SOX9 transcription via the the mevalonate pathway.Part ?: RhoA was highly actived in osteosarcoma cells,and by inhibiting RhoA activity and the assembly of cytoskeleton via down-regulating the mevalonate pathway,simvastatin intervention repressed the migration and invasion.Simvastatin regulated YAP activity and SOX9 transcription via RhoA and the assembly of cytoskeleton.Part ?: Reflected with low incidence of lung metastasis(20% versus80%),less lung metastatic nodules(0.2 nodules/mouse versus 1.6nodules/mouse),and prolonged survival time(P<0.005),simvastatin treatment displayed a positive effect on decreasing osteosarcoma-induced lung metastasis in in nude mice.Besides,the up-regulation of RhoA,YAP and SOX9 were also verified in osteosarcoma-induced lung metastatic nodules.Conclusions:RhoA,YAP and SOX9 are highly activated in osteosarcoma cells,promoting their migration and invasion.Simvastatin suppresses the migration and invasion of osteosarcoma cells by regulating the expression of SOX9 via the mevalue pathway.SOX9 is a direct target gene of YAP-TEAD,and simvastatin regulates SOX9 transcription by modulating YAP activity via RhoA and cytoskeleton.Moreover,simvastatin inhibits lung metastasis of osteosarcoma cells in vivo.Above all,simvastatin inhibits the metastasis of osteosarcoma by inhibiting RhoA activity and the assembly of cytoskeleton,reducing the YAP activity,and then down-regulating the YAP-mediated SOX9 transcription.