The Role And Molecular Mechanisms Of HSP90AA1 In Drug Resistance Of Osteosarcoma

[Background]Osteosarcoma is the most common primary bone tumor in children and adolescents.Treatment with a combination of neoadjuvant chemotherapy and surgery has improved the survival rate of osteosarcoma patients.Doxorubicin,cisplatin and methotrexate are commonly used chemotherapy drugs in osteosarcoma treatment.In the last three decades,osteosarcoma treatments often fail due to the development of chemoresistance and patients' poor respond to these drugs.Even though additional doses or drugs are used,these patients will not have good response to chemothsrapy and will still undergo local recurrence and metastasis,reducing the 5-year-survival rates to only 20%.For this poor prognosis,drug resistance is the main reason.Thus,to develop novel therapies and to finally improve the prognosis of osteosarcoma patients,it is very important to thoroughly understand the molecular mechanisms of the chemotherapy resistance occurred in osteosarcoma cells.Heat shock proteins are characterized as highly conserved chaperone proteins which play an important role in cell survival.It has been found that heat shock proteins are responsible for many cytoprotective mechanisms especially under stress conditions,such as anoxia,chemotherapy and acute injury.HSP90AA1 is one of the most important heat shock protein.The main content of our research is that whether HSP90AA1 gene is responsible for drug resistance in osteosarcoma.It has been reported that,except the cytoprotective effect,HSP90AA1 gene is also associated with invasion and migration of cancer cells.HSP90AA1 is regarded as essential for poor prognosis.MicroRNAs(miRNAs)are small non-coding single stranded RNAs.It has been found that abnormal expression of miRNAs is associated with development and progression of tumor.Accumulating evidence has indicated that miRNAs participate in a broad range of biological processes,such as proliferation,apoptosis,invasion and metastasis of different kind of tumors.Hence,which miRNA may diretly target HSP90AA1? What effects do they have on the malignant behavior of osteosarcoma? These are also the content of our research.[Aims]The goal of our study is to explore the molecular mechanism of drug resistance in osteosarcoma chemotherapy,find the therapeutic target and finally improve the survival rate of oeteosarcoma patients.We are aim to identify the miRNA which targets HSP90AA1 and to explore the role of them in the invasion and migration of osteosarcoma by using cell biology experiments.[Methods]shRNAs were transfected into osteosarcoma cells for knockdown of HSP90AA1 gene.Stable HSP90AA1 overexpressing osteosarcoma cell lines were obtained by lentivirus infection.mRNA and protein expressions of HSP90AA1 in osteosarcoma cells were tested by quantitative real-time PCR and western blot,respectively.The proliferation of osteosarcoma cell was texted by CCK-8 assay and the apoptosis in osteosarcoma cells was assessed using the Annexin V-PE/propidium iodide apoptosis detection kit by flow cytometric analysis.Autophagy of osteosarcoma cells was detected by western blot of LC3,transmission electron microscopy and fluorescence microscope.mRFP-GFP-LC3 lentiviral transfection was also performed to detect autophagic flux.NOD/SCID mices were inoculated with MG-63 tumor cells transfected with HSP90AA1 specific shRNA.TUNEL and LC3 staining were performed to detect apoptosis and autophagy of resected tumor tissues.Western blot was used to examine the proteins related to singnaling pathway,such as Akt,p-Akt,mTOR,p-mTOR,JNK,p-JNK,p38 and p-p38.The publicly available algorithms and luciferase reporter assay were used to predict potential miRNA which targets HSP90AA1.mRNA expressions of miR-495 in osteosarcoma cells and osteosarcoma tissues were tested by quantitative real-time PCR.miRNA-495 mimic,miRNA-495 inhibitor or HSP90AA1 shRNA were transfected into osteosarcoma cells,and then the wound-healing assays and matrigel invasion assays were performed to detected the osteosarcoma cells invasion and migration.The biomarker of epithelial-mesenchymal transition,such as E-cadherin,Vimentin,Snail1 and ZEB1,were examined by quantitative real-time PCR and western blot.Stable miR-495 overexpressing osteosarcoma cell lines were obtained by lentivirus infection.NOD/SCID mices were injected with miR-495 overexpressing osteosarcoma cells through the tail vein.Individual organs from the mice were removed,and metastatic tissues(lung)were analyzed with H&E staining.[Results]Doxorubicin,cisplatin,and methotrexate,which are commonly used in chemotherapy,each induced HSP90AA1 upregulation in human osteosarcoma cells.Overexpression of HSP90AA1 enhanced the cellular apoptosis and inhibited cell proliferation to cisplatin,docetaxel and methotrexate,indicating drug resistance in osteosarcoma cells.Suppression of HSP90AA1 restored the sensitivity of osteosarcoma cells to chemotherapy both in vivo and in vitro.Mechanism study indicated that autophagy is responsible for the chemoresistance in osteosarcoma cells.HSP90AA1 increased drug resistance by inducing autophagy and inhibiting apoptosis.Suppression of HSP90AA1 diminished autophagic protection in response to chemotherapy in osteosarcoma cells.Moreover,HSP90AA1 promotes autophagy through PI3K/Akt/mTOR pathway and inhibits apoptosis through JNK/P38 pathway.We demonstrated that HSP90AA1 is a direct target of miR-495 using luciferase reporter assay and Western blot analysis.In this study,we firstly found that miR-495 is markedly downregulated in both osteosarcoma tissues and cell lines.When the exogenous miR-495 was introduced,osteosarcoma cells showed decreased capability of migration and invasion with decreased Snail1,ZEB1,vimentin and increased E-cadherin.when the endogenous miR-495 was inhibited,osteosarcoma cells presented increased migration and invasion ability with increased Snail1,ZEB1,vimentin and decreased E-cadherin.Function of miR-495 in vivo was also examined in mice xenograft model and we found it significantly inhibiting the lung-metastasis of osteosarcoma cells.Furthermore,knockdown of HSP90AA1 can restore the increased cell invasion and migration in miR-495-knockdown cells and over expression of HSP90AA1 can neutralize the impaired metastatic ability of miR-495 mimic-treated cells.[Conclusion]We showed that chemotherapy agents can induce HSP90AA1 expression in osteosarcoma cells.And HSP90AA1,acting as an important regulator of autophagy,is a critical factor in the development of osteosarcoma chemoresistance both in vitro and in vivo.HSP90AA1 provides a novel therapeutic target for improving osteosarcoma treatment.Our findings proved for the first time that miR-495 acts as a tumor suppressor in osteosarcoma and inhibits cell migration and invasion by targeting HSP90AA1.miR-495 suppresses the invasion and metastasis of osteosarcoma cells through inhibiting epithelial to mesenchymal transition.miR-495 may be a promising therapeutic target for osteosarcoma treatment.