In order to examine the underlying relationship between OS cells and EMT, we silenced ATG5, which is recognized to have an essential role in autophagosome formation. transcription-polymerase chain reaction (RT-PCR), miR-210-5p was found to be upregulated in clinical OS specimens and cell lines. Further functional analysis demonstrated that miR-210-5p promoted epithelialCmesenchymal transition (EMT) and induced oncogenic autophagy. Luciferase reporter assay, RNA-ChIP, and western blot analysis confirmed that PIK3R5, an essential regulator in the AKT/mTOR signaling pathway, is a target downstream gene of miR-210-5p. Overexpression or knockdown of PIK3R5 reversed the functional role of overexpression or knockdown of miR-210-5p, respectively. Silencing autophagy-related gene 5 (ATG5) abolished the functional effects of miR-210-5p upregulation or PIK3R5 knockdown in OS cells. In vivo, miR-210-5p overexpression promoted OS tumor growth and pulmonary metastasis. Taken together, our results demonstrated that miR-210-5p promoted EMT and oncogenic autophagy by suppressing the expression of PIK3R5 and regulating the AKT/mTOR signaling pathway. Therefore, inhibition of miR-210-5p may represent a promising treatment for OS. test was used to compare two groups. Statistical analyses were performed using SPSS v. 22.0 (SPSS Inc., Chicago, IL, USA). em P /em ? ?0.05 was considered statistically significant. Results Upregulation of miR-210-5p in clinical OS specimens and cell lines First, we assessed the expression of miR-210-5p in 62 paired OS specimens and matched adjacent normal specimens. It was found the expression level of miR-210-5p was significantly upregulated in OS tissues compared with adjacent normal tissues (Fig. ?(Fig.1a).1a). FISH was then used to detect the miR-210-5p expression level, and the results shown in Fig. ?Fig.1b1b confirmed the above RT-PCR results. It was also found that miR-210-5p expression was higher in the metastasis group compared with the non-metastasis group (Fig. ?(Fig.1c).1c). The representative radiological images of OS patients with or without pulmonary metastasis are shown in Supplementary Fig. S1. In OS cell lines, including HOS, Saos-2, SW1353, U2OS, and MG63, the expression level of miR-210-5p was upregulated in OS cell lines when compared with the normal human osteoblast cell line hFOB 1.19 (Fig. ?(Fig.1d).1d). In addition, the expression level of miR-210-5p was obtained from the GEO online database and confirmed that the expression of miR-210-5p was higher in OS cell lines (Supplementary Fig. S2A). Furthermore, we analyzed the relationship between the expression level of miR-210-5p and the clinicopathological characteristics in Obatoclax mesylate (GX15-070) OS patients (Supplementary Table S1). The expression level of miR-210-5p was found to be significantly positively correlated with TNM stage, lung metastasis, and tumor size. Open in a separate window Fig. 1 miR-210-5p is upregulated in clinical OS specimens and cell lines.a The expression of miR-210-5p in 62 pairs of clinical OS specimens and matched adjacent normal tissues. b Representative FISH images of miR-210-5p in clinical OS specimens and matched adjacent normal tissues. Scale bar?=?50?m. c The expression of miR-210-5p in the metastasis group compared with the non-metastasis group. d The relative expression of miR-210-5p in OS cells Obatoclax mesylate (GX15-070) and the hFOB 1.19 cell line. e, f The expression of miR-210-5p in HOS and MG63 cells transfected with LV-miR-210-5p or ANTI-miR-210-5p. miR-210-5p promotes tumor invasion and migration in OS cells Based on the expression level of miR-210-5p in the OS cell lines, HOS and MG63 cell lines were transfected with LV-miR-210-5p or ANTI-miR-210-5p lentivirus, respectively. The expression level after transfection was assessed using miRNA RT-PCR (Fig. 1e, f). Gene set enrichment analysis (GSEA) was performed, and it was found that miR-210-5p expression was positively correlated with EMT-associated gene signatures, which means that miR-210-5p may have an impact on the EMT process in OS (Fig. ?(Fig.2a).2a). Staining of vimentin, a mesenchymal biomarker, showed that the expression level of vimentin was higher in the high miR-210-5p group (Fig. Obatoclax mesylate (GX15-070) ?(Fig.2b).2b). Furthermore, overexpression of miR-210-5p in HOS cells increased the expression levels of mesenchymal markers including N-cadherin, Vimentin, and MMP2, but decreased the expression of epithelial cell marker E-cadherin. In contrast, suppression of miR-210-5p in MG63 cells showed the opposite effects (Fig. ?(Fig.2c).2c). A transwell invasion assay was then conducted to investigate the impact of miR-210-5p on cell invasion and migration Mouse monoclonal to CD95(Biotin) ability in OS. As shown in Fig. ?Fig.2d,2d, overexpression of miR-210-5p significantly promoted HOS cell invasiveness, and silencing miR-210-5p attenuated MG63 cell invasiveness (Fig..