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Osteosarcoma (OS) is the most common primary malignant tumor of bone. The aim of this study was to investigate the regulatory mechanisms of M2 macrophage exosomes (M2-Exos) in ferroptosis in OS. A mouse model was established to investigate the in vivo role of M2-Exos. We investigated their effects on ferroptosis in OS using erastin, a ferroptosis activator, and deferoxamine mesylate, an iron chelator. In vitro, we investigated whether the Apoc1/Acyl-CoA Synthetase Family Member 2 (ACSF2) axis mediates these effects, using shApoc1 and shACSF2. The mechanisms whereby Apoc1 regulates ACSF2 were examined using cyclohexanone, a protein synthesis inhibitor, and MG132, a proteasomal inhibitor. M2-Exos reversed the inhibitory effects of erastin on OS cells, thus enhancing their viability, migration, invasion, proliferation, and reducing ferroptosis. Apoc1 was highly expressed in M2-Exos, and interfering with this expression reversed the effects of M2-Exos on OS cells. ACSF2 mediated the effects of M2-Exos-derived Apoc1. Apoc1 interacted with ACSF2, which, in turn, interacted with USP40. Apoc1 overexpression increased ACSF2 ubiquitination, promoting its degradation, whereas USP40 overexpression inhibited ACSF2 ubiquitination and promoted its expression. Apoc1 overexpression inhibited ACSF2 binding to USP40. M2-Exos-derived Apoc1 promoted ferroptosis resistance by inhibiting USP40 binding to ACSF2 and promoting ACSF2 ubiquitination and degradation, thus enhancing OS development., (© 2024 Wiley Periodicals LLC.)

A growing amount of evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in osteosarcoma (OS). However, little knowledge is available about the functional roles and molecular mechanisms of lncRNA Alu-mediated p21 transcriptional regulator (APTR) in OS. Herein, APTR expression was demonstrated to be significantly upregulated in OS tumor tissues and four OS cell lines (including MG63, 143B, Saos-2, and HOS) compared with the adjacent tissues and human osteoblast cell line hFOB1.19, respectively. We confirmed miR-132-3p to be a target for APTR, and its expression was demonstrated to be inhibited by APTR. In functional terms, knockdown of APTR and overexpression of miR-132-3p both, remarkably repressed human OS cell proliferation, invasion and migration, and induced apoptosis. Also, Yes-associated protein 1 (YAP1) was determined as an inhibitory target of miR-132-3p. Moreover, our findings demonstrated that the repression of YAP1 protein expression and the suppression of Ki-67, MMP9, and Bcl2 expression induced by APTR knockdown required increased miR-132-3p. Thus, APTR contributed to OS progression through repression of miR-132-3p and upregulation of YAP1 expression. Therefore, we have uncovered a novel regulatory mechanism by which the APTR/miR-132-3p/YAP1 axis can regulate OS progression., (© 2018 Wiley Periodicals, Inc.)

Diallyl disulfide (DADS), a volatile component of garlic oil, exerts anticancer activity in various types of cancers, while its anticancer effects against osteosarcoma (OS) have not been previously explored. This study aimed to investigate the anticancer potential of DADS in OS and to explore the underlying mechanisms. DADS reduced the cell viability and increased the expression of miR-134 in OS cell lines, and this effect was in a time- and concentration-dependent manner. Furthermore, in vitro functional assays revealed that DADS significantly inhibited the proliferation and invasion of human OS U2OS and MG-63 cells, which was partially reversed by miR-134 inhibitor transfection. DADS exhibited in vivo antitumor activity and upregulated miR-134 expression in xenograft tumors. Downregulation of miR-134 attenuated DADS-induced antitumor capacity. Further bioinformatics prediction analysis revealed that the 3'-untranslated region (3'-UTR) of Forkhead Box M1 (FOXM1) harbored miR-134-binding sites, and overexpression of miR-134 repressed the luciferase activity of the reporting vector containing FOXM1 3'-UTR. Both miR-134 overexpression and DADS inhibited FOXM1 expression in U2OS cells, while enforced expression of FOXM1 suppressed DADS-induced antiproliferation and anti-invasion capacity in U2OS cells. Furthermore, DADS treatment led to significant downregulation of cyclin D1, c-myc, and lymphoid enhancer-binding factor 1 expression, but the remarkably upregulated p21 level in U2OS cells. Collectively, DADS could be a promising anticancer agent for OS, and the underlying mechanisms might be associated with the antiproliferation and anti-invasion properties through upregulating miR-134 expression., (© 2018 Wiley Periodicals, Inc.)

Osteosarcoma (OS) is a common primary malignant bone tumor in young adolescents. About 30% of patients with OS have a recurrence, and the overall survival after OS recurrence is not good. In this study, we aimed to analyze and identify factors that influence prognosis after OS relapse. We retrieved the Gene Expression Omnibus data set and collected a series of transcriptome data with clinical information, including microRNA (miRNA) and messenger RNA (mRNA) expression profiles in recurrent OS. Upon comparison of the dysregulated genes of survival status in the recurrent OS samples, it was found that there were 268 differential expressed (DE) mRNAs and six DE miRNAs. These genes are related to pathways in cancer. We also predicted the interaction networks of these DE mRNAs and miRNAs. Further, we applied cibersort to estimate the proportion of immune cell types and we discovered that natural killer cells and macrophages have different abundance between good prognosis and poor prognosis. Our study indicates that for recurrent OS samples, there are several differences between these two groups, including gene expression and the status of immune activation. The immunity status is a candidate signature for disease prediction, prevention, and therapy choices., (© 2018 Wiley Periodicals, Inc.)

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS., (© 2018 Wiley Periodicals, Inc.)

Osteosarcoma (OS) is a primary malignant bone tumor with high morbidity. Developing new therapeutic approaches with neoadjuvant is of great interest in OS treatment. Reportedly, ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and radiation resistance gene 3 related (ATR)-p53 signaling is considered as a critical DNA damage signaling pathway sensitizing cancer cells to chemotherapies; while wild-type p53-induced phosphatase 1 (WIP1), an oncogene overexpressed in diverse cancers, has been regarded as a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Herein, the expression of WIP1 in OS tissues and cell lines was examined; to investigate the mechanism of WIP1 abnormal upregulation, online tools were used to predict the upstream regulatory microRNAs (miRNAs) targeting WIP1. Among the candidate miRNAs, the expression and detailed function of miR-590 were validated. Through binding to the 3'-untranslated region of WIP1, miR-590 inhibited WIP1 expression and, therefore, enhanced the effect of Dox on OS cell proliferation and apoptosis through downstream ATM-p53 signaling. Moreover, RELA could bind to the promoter region of miR-590 to inhibit its expression, thereby affecting downstream WIP1 and ATM-p53 signaling. The expression of p65 was upregulated in OS tissues, indicating that the effect of p65 inhibition on cell viability, apoptosis, and related mechanisms could be partially restored by miR-590 inhibition. Taken together, these results showed that p65-mediated miR-590/WIP1/ATM-p53 modulation might be a novel target to enhance the cellular effect of Dox on OS cell lines., (© 2018 Wiley Periodicals, Inc.)

Osteosarcoma (OS), as the most frequent primary malignancy of bone, is characterized by the presence of malignant mesenchymal cells. In the current study, our aim was to explore the possible effects Fos-like antigen-1 (FOSL1) had on the silencing regarding OS cell proliferation, invasion, and migration through the activation of the extracellular-signal-regulated kinase (ERK)/activator protein-1 (AP-1) signaling pathway. After the collection of OS on top of already having the adjacent normal tissue samples, the protein positive expression rate of FOSL1 was then measured by implementing the use of immunohistochemistry and discovered that FOSL1 was robustly expressed in OS. Later, to better grasp the impact FOSL1 projects on OS and its underlying mechanism, we determined the OS related genes as well as the ERK/AP-1 signaling pathway related genes expression by using a reverse-transcription quantitative polymerase chain reaction and western blot assay techniques. The results of the aforementioned two experiments revealed that the FOSL1 depletion had downregulated the expression of OS related genes by simultaneously downregulating the ERK/AP-1 signaling pathway. Moreover, cell proliferation, cycle, apoptosis, invasion, and migration of FOS1 were all tested by using a cell counting kit-8 assay, flow cytometry, Transwell assay, and scratch test, and these results presented that silencing of the FOSL1 gene inhibited OS cell proliferation, invasion, and migration. Our findings revealed a novel mechanism by which FOSL1 depletion played a significantly negative role in the OS progression through the regulation of the ERK/AP-1 signaling pathway. Functional suppression of FOSL1 might be a future therapeutic strategy regarding OS., (© 2018 Wiley Periodicals, Inc.)

Osteosarcoma (OS) is considered the most common type of primary malignant bone tumor, which has a high rate of mortality in children and adolescents. However, the current treatment methods for OS are ineffective. Therefore, there is an urgent requirement to identify the critical targets. This study aimed to identify the roles and significance of microRNA-216b (miR-216b) in OS. To explore the cellular and molecular functions of miR-216b and Forkhead Box M1 (FoxM1) in OS, the expression of miR-216b and FoxM1 at the transcriptional level was measured using quantitative real-time PCR (qRT-PCR). Wound healing assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay (MTT) assay, flow cytometry, and transwell invasion assay were conducted to study the function of miR-216b and FoxM1 in OS cells. Dual luciferase reporter assay was performed to identify the relationships between miR-216b and FoxM1. qRT-PCR results revealed that miR-216b expression was significantly downregulated, and FoxM1 was observed to be significantly upregulated in human OS cell lines (MG-63) and tissues. MTT data showed that upregulation of miR-216b expression led to cell growth inhibition in MG-63 cells. The results of the invasion assay and wound healing assay illustrated that miR-216b upregulation or FoxM1 downregulation could inhibit the invasion and migration in MG-63 cells. In vivo, the tumor volume was significantly decreased by miR-194 mimic treatment compared with the control group. Furthermore, the results of the luciferase assay indicated that FoxM1 is a direct target of miR-216b. These findings may provide novel insights into the molecular mechanism of miR-216b and FoxM1 in the progression of OS, and suggested that miR-216b may serve as a potential tumor inhibitor of OS by targeting FoxM1., (© 2018 Wiley Periodicals, Inc.)

Increasing evidence shows that the long noncoding RNA nuclear enriched abundant transcript 1 (NEAT1) plays important roles in tumor progression. However, the function and the underlying mechanism of NEAT1 in osteosarcoma (OS) remain unclear. In the present study, we found that NEAT1 expression was significantly upregulated in OS tissues and cell lines. High NEAT1 expression was closely associated with advanced clinicopathologic features and poor overall survival of patients with OS. Using in vitro function assay, we found that NEAT1 could promote the proliferation, invasion, and epithelial-mesenchymal transition (EMT) process of OS cells. NEAT1 could also promote OS cell growth in vivo. In addition, our studies showed that miR-186-5p was a downstream target of NEAT1 in OS. Functionally, miR-186-5p suppressed the proliferation, invasion, and EMT process of OS cells. Furthermore, our data revealed that HIF-1α was a downstream target of miR-186-5p and that NEAT1 could exert its tumor oncogenic roles on OS cells via the miR-186-5p/HIF-1α axis. Taking our results together, we elucidated that the NEAT1/miR-186-5p/HIF-1α axis might be a therapeutic approach for the treatment of OS., (© 2018 Wiley Periodicals, Inc.)

Overcoming platinum drug resistance represents a major clinical challenge in osteosarcoma (OS) treatment. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Notch signaling is implicated in regulating CSCs and tumor resistance to platinum. Thus, we attempt to investigate whether inhibiting of Notch pathway could sensitize cisplatin (CDDP) to CDDP-resistant OS cells and the underlying molecular mechanisms. OS cell lines resistant to CDDP were treated with DAPT, CDDP or combination, we present evidences that DAPT enhances the cytotoxic effect of CDDP in resistant OS by inhibiting proliferation, resulting in G0/G1 cell-cycle arrest, inducing apoptosis, and reducing motility. In addition, DAPT targeting depletes OS stem cells (OSCs), thus increasing tumor sensitivity to platinum, which indicating that a dual combination targeting both OSCs and the bulk of tumor cells are needed for tumor eradication. We also found that the combination of CDDP and DAPT exhibit additive suppression on phosphorylated AKT and ERK, contributing to the anti-cancer effects. In animal model, this combination therapy inhibits the growth and metastasis of CDDP resistant tumor xenografts in nude mice to a greater extent than treatment with either reagent alone. Based on these results, we conclude that CDDP plus DAPT was able to sensitize CDDP-resistant human OS cells to CDDP by downregulation of Notch signaling. CDDP and DAPT combination treatment may be effective and promising for advanced OS., (© 2018 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.)

Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of osteosarcoma (OS), and is associated with resistance to therapy, poor survival, and a malignant phenotype. The purpose of the present study was to investigate the role and underlying mechanism of SUMO-specific protease 1 (SENP1)/hypoxia-inducible factor-1α (HIF-1α) feedback loop in hypoxic microenvironment of OS. We observed that the expression of SENP1 was remarkably upregulated in OS cells. Additionally, there was a concomitant high expression of HIF-1α and SENP1 in MG-63 cells under a hypoxic microenvironment. Interestingly, blockage of HIF-1α repressed the enhancement of SENP1. Moreover, knockdown of SENP1 with siRNA notably inhibited cell viability and accelerated cell apoptosis accompanied by a decrease in the expression of Bcl-2 and an increase in the expression of Bax in MG-63 cells following exposure to hypoxia. Furthermore, repression of SENP1 dramatically suppressed cell invasive ability through modulating epithelial-mesenchymal transition (EMT) marked genes as reflected by the upregulation of E-cadherin, as well as the downregulation of vimentin and N-cadherin under hypoxic conditions. Most importantly, SENP1 positively regulated HIF-1α expression level in the setting of hypoxic; subsequently, depletion of SENP1 expression markedly ameliorated vascular endothelial growth factor (VEGF) production triggered by hypoxia. Taken together, positive feedback loop between HIF-1α and SENP1 in the regulating of the process of cell proliferation, invasion, and EMT in OS cells under hypoxic conditions, suggesting that the SENP1/HIF-1α axis may serve as a new potential therapeutic agent for OS treatments., (© 2017 Wiley Periodicals, Inc.)

MiR-214 belongs to a family of microRNA (small, highly conserved noncoding RNA molecules) precursors that play a pivotal role in biological functions, such as cellular function, tissue development, tissue homeostasis, and pathogenesis of diseases. Recently, miR-214 emerged as a critical regulator of musculoskeletal metabolism. Specifically, miR-214 can mediate skeletal muscle myogenesis and vascular smooth muscle cell proliferation, migration, and differentiation. MiR-214 also modulates osteoblast function by targeting specific molecular pathways and the expression of various osteoblast-related genes; promotes osteoclast activity by targeting phosphatase and tensin homolog (Pten); and mediates osteoclast-osteoblast intercellular crosstalk via an exosomal miRNA paracrine mechanism. Importantly, dysregulation in miR-214 expression is associated with pathological bone conditions such as osteoporosis, osteosarcoma, multiple myeloma, and osteolytic bone metastasis of breast cancer. This review discusses the cellular targets of miR-214 in bone, the molecular mechanisms governing the activities of miR-214 in the musculoskeletal system, and the putative role of miR-214 in skeletal diseases. Understanding the biology of miR-214 could potentially lead to the development of miR-214 as a possible biomarker and a therapeutic target for musculoskeletal diseases., (© 2018 Wiley Periodicals, Inc.)