Background: miRNAs are confirmed to play essential roles in tumorigenesis and progression of cancers, including Ewing’s sarcoma. miR-185 has been reported to be downregulated in some tumors, whereas the role of miR-185 in Ewing’s sarcoma remains unclear.
Purpose: The objective of this study was to investigate the role of miR-185 in the progression and metastasis of Ewing’s sarcoma and explore the associated mechanism.
Materials and methods: Ewing’s sarcoma cell line RD-ES was transfected with pCMV-MIR-miR185 vector to upregulate the expression of miR-185. Cell Counting Kit 8 and colony formation assays were used to assess the effect of miR-185 on cell proliferation. The effect of miR-185 on cell migration and invasion was detected by transwell assay. Flow cytometry assay was performed to detect apoptosis rate of RD-ES cells. The protein levels of apoptosis-related proteins was determined using Western blot assay or immunohistochemistry assay. Dual-luciferase reporter assay was used to validate the regulation between miR-185 and its target gene.
Results: Upregulation of miR-185 caused significant inhibition on cell growth capacity, migration and invasion of Ewing’s sarcoma cell RD-ES. Besides, upregulation of miR-185 was observed to accelerate cell apoptosis in a mitochondrial pathway through regulating Bcl-2/Bax, Caspase 3, and Caspase 9 in Ewing’s sarcoma in vitro . Moreover, upregulation of miR-185 was found to suppress the PI3K/Akt/mTOR and Wnt/β-catenin pathways in RD-ES cells. Furthermore, we identified that E2F6 was a target gene for miR-185, and the suppression on cell proliferation caused by overexpression of miR-185 was significantly rescued by the upregulation of E2F6 in RD-ES cells. 
Conclusion: miR-185 is involved in cell growth, motility and survival of Ewing’s sarcoma as a tumor suppressor via suppressing PI3K/Akt/mTOR and Wnt/β-catenin pathways and targeting E2F6.
Keywords: miR-185, PI3K/Akt/mTOR pathway, Wnt/β-catenin pathway, E2F6, Ewing’s sarcoma