Effects And Mechanism Of Hypoxia-induced MiR-378a-3p On Invasion And Epithelial-to-Mesenchymal Transition Of Osteosarcoma Via Regulating BYSL

Objective This study aims to reveal the effect of miR-378a-3p on osteosarcoma metastasis by targeting BYSL under hypoxic microenvironment,and explore their regulatory mechanisms and therapeutic significance in osteosarcoma.Methods We initially evaluated BYSL transcription levels and prognostic role in osteosarcoma using The Cancer Genome Atlas(TCGA)and the Gene Expression Omnibus(GEO)databases.Paraffin-embedded biopsy specimens from 51 patients with non-pretreated osteosarcoma in The First Affiliated Hospital of Guangxi Medical University from March 2011 to December 2019 were studied in a retrospective immunohistochemical study.Kaplan-Meier survival and Cox regression analysis were used to assess the clinical role of BYSL using immunohistochemistry and available follow-up data.Next,the potential BYSL-targeting miRNA was explored using bioinformatics and luciferase reporter assay.The m RNA levels of miR-378a-3p and BYSL were detected under normoxia and hypoxia.Loss-of-function and Gain-of-function analyses were performed to investigate the role of miR-378a-3p and BYSL in cell invasion,migration,apoptosis,and EMT on osteosarcoma under normoxia and hypoxia,respectively.Furthermore,the function of miR-378a-3p on osteosarcoma cell growth in vivo was investigated in a nude mice xenograft model.At last,the regulatory mechanisms among hypoxia,miR-378a-3p,BYSL,and Nrf-2 pathways were assessed.Results In this study,BYSL was found significantly up-regulated in osteosarcoma samples and closely correlated with clinical prognosis in osteosarcoma patients using bioinformatics analysis.In our in-house cohort,high expression of BYSL was positively related to the metastasis status and poor prognosis using immunohistochemical analysis combined with survival follow-up data.Cox regression analysis indicated that BYSL was an independent risk factor for the overall survival rate of patients with osteosarcoma.Using dual-luciferase reporter assays,we showed that miR-378a-3p regulated BYSL expression by directly binding to the 3’-UTR of BYSL.The results of q RT-PCR showed that hypoxia increased BYSL expression and reduced that of miR-378a-3p.Therefore,overexpression or inhibition of miR-378a-3p was performed to investigate the role of miR-378a-3p.Loss-of-function analyses revealed that miR-378a-3p inhibited osteosarcoma invasion and epithelial-to-mesenchymal transition via BYSL regulation under normoxia,and gain-of-function analyses also revealed that miR-378a-3p inhibited osteosarcoma invasion and epithelial-to-mesenchymal transition via BYSL regulation under hypoxia.Moreover,overexpression of miR-378a-3p markedly suppressed the tumor growth in vivo.At last,we found that hypoxia enhanced Nrf-2 localization within the nucleus and miR-378a-3p regulated osteosarcoma metastasis by targeting BYSL and activating Nrf-2.Conclusion Our results reveal that BYSL could be served as an independent risk factor for predicting the prognosis of osteosarcoma patients,and hypoxia/miR-378a-3p/BYSL/Nrf-2 may play an important role in regulating metastasis of osteosarcoma.