RESUMO
Circular RNAs (circRNAs) have been demonstrated to be involved in osteosarcoma (OS) development; however, the underlying mechanism of circKMT2D in OS progression remains unclear. The present study aimed to elucidate how circKMT2D could affect hydrogen peroxide (H2O2)-induced OS progression. H2O2 (100 µmol/l) was used to treat MG63 and U2OS cells. The cell viability, invasive ability, apoptosis and circKMT2D expression were detected using Cell Counting Kit-8 assay, Transwell assay, flow cytometry and reverse transcription-quantitative PCR, respectively. Furthermore, MG63 and U2OS cells transfected with circKMT2D short hairpin RNA and negative control were treated with H2O2, and circKMT2D expression and cell phenotype were determined. Dual-luciferase reporter assay was conducted to determine the association between circKMT2D and miR-210 expression level. Rescue experiments were conducted to examine the mechanisms through which circKMT2D and miR-210 could affect H2O2-treated MG63 cells. In addition, the effects of miR-210 on the expression of the autophagy-related proteins Beclin1 and p62 in H2O2-treated MG63 cells were detected by western blotting. An autophagy inhibitor was used to treat the MG63 cells, and whether miR-210 could affect the H2O2-treated MG63 cell phenotype through autophagy was investigated. The results demonstrated that H2O2 treatment promoted cell apoptosis and decreased cell viability, invasive ability and circKMT2D expression in MG63 and U2OS cells. Furthermore, circKMT2D knockdown decreased the cell viability and invasive ability and enhanced the apoptosis of H2O2-treated MG63 and U2OS cells. circKMT2D possessed binding sites for miR-210 and inhibited miR-210 expression. In H2O2-treated MG63 cells, miR-210 silencing partially reversed the circKMT2D knockdown-induced cell viability inhibition and apoptosis promotion. In addition, miR-210 elevated Beclin1 expression and decreased p62 expression in H2O2-treated MG63 cells. The use of the autophagy inhibitor partially reversed the miR-210 overexpression-induced promotion of apoptosis and inhibition of the viability and invasive ability of H2O2-treated MG63 cells. Taken together, these findings indicated that circKMT2D knockdown may contribute to the inhibition of H2O2-attenuated OS progression via miR-210/autophagy pathway.
RESUMO
Osteosarcoma (OS) cells are one of the primary cancer-related causes of death around the world. Long noncoding RNAs are key for OS progression; however, the detailed molecular mechanism remains unknown. LINC00657, miR-106a, and programmed death ligand-1 (PD-L1) genes expression were detected by reverse transcription-quantitative PCR (RT-qPCR) and Western blot approaches. Invasion and lymphangiogenesis were studied using transwell invasion assay and lymphatic vessel formation assay, respectively. We used bioinformatic analyses to identify putative targets of LINC00657 and miR-106a. Luciferase activity was measured by dual-luciferase reporter assay. PD-L1 protein levels were examined by flow cytometry experiments. LINC00657 knockdown attenuates cell invasion and tumor growth of MG63 and lymphatic vessel formation. miR-106a directly binds LINC00657 and they regulate each other. Furthermore, miR-106a inhibitor strikingly enhanced lymphatic vessel formation and invasion of shLINC00657 MG63 cells. miR-106a mimic directly targeted and downregulated PD-L1. PD-L1 overexpression largely rescued miR-106a mimic-modulated OS cell metastasis. LINC00657 and PD-L1 were upregulated in clinical OS tumors compared to normal tissues. Lower expression levels of LINC00657 and PD-L1 were closely associated with higher overall survival of patients with OS. Here, we suggest a novel mechanism for LINC00657-regulated metastasis of OS cells by regulating the miR-106a/PD-L1 axis. Our conclusions facilitate the development of therapeutical approaches by targeting LINC00657.
