Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. The lncRNA growth arrest-specific 5 (GAS5) is reported to be a tumor suppressor in multiple cancers. However, the roles of GAS5 and its related miRNAs in osteosarcoma are poorly understood. This study explored the potential functions and mechanisms of GAS5 in the tumorigenesis of osteosarcoma. Here, the expression of GAS5, miR-221 and aplasia Ras homologue member I (ARHI) was determined in osteosarcoma tissues and cells by Real-time PCR (RT-qPCR). The underlying mechanism of GAS5 in osteosarcoma growth was analyzed via MTT, Transwell, RT-qPCR, Western blot, dual-luciferase reporter assay, RNA immunoprecipitation, and xenograft models after GAS5 overexpression. GAS5 and ARHI levels were significantly reduced, while miR-221 increased, both in osteosarcoma tissues and cells. Overexpression of GAS5 suppressed the proliferation, migration, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. GAS5 could directly bind to miR-221 to decrease miR-221 expression and enhance ARHI expression. The effect of GAS5 overexpression on the proliferation, migration and EMT was reversed by miR-221 mimics or ARHI siRNA in osteosarcoma cells. Additionally, GAS5 suppressed tumor volume, Ki-67 and PCNA staining, and EMT process in the development of osteosarcoma in vivo. Taken together, lncRNA GAS5 functions as a competing endogenous RNA for miR-221 to suppress cell growth and EMT in osteosarcoma by regulating the miR-221/ARHI pathway. J. Cell. Biochem. 118: 4772-4781, 2017. © 2017 Wiley Periodicals, Inc.

Bone defects resulting from non-union fractures or tumour resections are common clinical problems. Long non-coding RNAs (lncRNAs) are reported to play vital roles in stem cell differentiation. The aim of this study was to elucidate the role of lncRNA-H19 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). Following the establishment of an osteogenic differentiation model in rats, the expression of H19, microRNA-149 (miR-149) and stromal cell-derived factor-1 (SDF-1) was measured by RT-qPCR. Thereafter, BMMSCs were isolated from rats and treated with a series of mimic, inhibitor or siRNA. SDF-1 expression, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were detected. The mineralized and calcified nodules were assessed by alizarin red S and Von Kossa staining.BMMSC surface markers were detected by flow cytometry. Western blot analysis was used to measure the expression of ALP, OCN, runt-related transcription factor 2 (RUNX2) and osterix (OSX) proteins. Lastly, dual-luciferase reporter gene assay and RNA immunoprecipitation were applied to verify the relationship of H19, miR-149 and SDF-1. Overexpressed H19 and SDF-1 and poorly expressed miR-149 were found in rats with osteogenic differentiation. H19 increased SDF-1 expression by binding to miR-149. H19 enhanced ALP activity, OCN content, calcium deposit and ALP, OCN, RUNX2 and OSX protein expression of BMMSCS by up-regulating SDF-1 via binding to miR-149. Taken together, up-regulated H19 could promote the osteogenic differentiation of BMMSCs by increasing SDF-1 via miR-149.

The incidence of osteosarcoma (OS) is increasing year by year, and the prognosis of patients with advanced OS is extremely poor due to the tendency of recurrence and chemotherapy resistance after surgery. Ferroptosis is a novel form of programmed cell death (PCD) that kills cells through iron-dependent lipid peroxidation. Current studies have shown that ferroptosis is closely related to OS and could reduce chemotherapy resistance to a certain extent, which has great therapeutic potential. In this paper, we review the regulatory mechanism of ferroptosis and its research progress in OS, hoping to provide new help for the clinical treatment of OS.

ARHI is an imprinted tumor suppressor gene and its methylation suppresses ARHI transcription levels to cause the development and progression of malignant tumors. Zebularine exerts a demethylation function for tumor suppressor genes. Our study aims to investigate the effect and mechanism of action of zebularine on the epigenetic modification of the ARHI gene, and whether this effect may modulate the viability and apoptosis of human osteosarcoma cells. We found that zebularine inhibited the viability and promoted apoptosis in osteosarcoma cells. Zebularine potentiated the expression of ARHI at both the protein and mRNA level. This was related to the downregulation of methylation of ARHI caused by zebularine. Zebularine suppressed the interaction of DNA methyltransferase 1 (DNMT1) with histone methyltransferase G9a, but had no effect on G9a alone. Knockdown of DNMT1 or G9a can induce a reduction of ARHI methylation. Therefore, we inferred that zebularine was likely to directly repress DNMT1 alone, but G9a was necessary to regulate the function of DNMT1 on ARHI methylation. Moreover, knockdown of ARHI rescued cell viability and apoptosis under the zebularine‐treated condition. We showed that zebularine inhibited viability and promoted apoptosis by disturbing the interaction between DNMT1 and G9a, thereby resulting in lower ARHI methylation and elevated ARHI expression in osteosarcoma cells.

Aplasia Ras homologue member Ⅰ (ARHI), an imprinted tumor-suppressor gene, is downregulated in various types of cancer. However, the expression, function and specific mechanisms of ARHI in human osteosarcoma (OS) cells remain unclear. The aim of the present study was to assess the effect of ARHI on OS cell proliferation and apoptosis and its associated mechanism. In the study, ARHI mRNA and protein levels were markedly downregulated in OS cells compared with the human osteoblast precursor cell line hFOB1.19. By generating stable transfectants, ARHI was overexpressed in OS cells that had low levels of ARHI. Overexpression of ARHI inhibited cell viability and proliferation and induced apoptosis. However, caspase-3 activity was not changed by ARHI overexpression. In addition, phosphorylated Akt protein expression decreased in the ARHI overexpression group compared to that in the control vector group. The knockdown of ARHI also resulted in the promotion of cell proliferation and the attenuation of apoptosis in MG-63 cells. Additionally, ARHI silencing increased the level of p-Akt. The present results indicate that ARHI inhibits OS cell proliferation and may have a key role in the development of OS.