Tumor Progression In Osteosarcoma Research Articles

RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway

BackgroundRab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism.MethodsTumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed.ResultsOverexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial–mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway.ConclusionOur study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.

Abstract 3803: The landscape of circular RNAs in metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the most common gastrointestinal malignancy and a leading cause of cancer death in the U.S.. Approximately 50% of patients with CRC develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs), covalently closed RNA molecules produced from pre-mRNAs through backsplicing, have recently emerged as an important class of non-coding RNAs regulating CRC tumorigenesis and progression. In this study we established a comprehensive catalog of circRNAs in mCRC. To evaluate circRNAs in mCRC progression, total RNA-Seq was performed on 30 matched normal, primary, and distant metastatic samples from 14 mCRC patients. Additionally, the transcriptome of five CRC cell lines were sequenced to evaluate circRNAs expressed in cell lines. CircRNAs were identified and quantified using the number of backspliced reads, normalized against the total number of mapped reads. Differential expression (DE) analysis was performed to identify circRNAs deregulated between normal, primary, and metastatic tissues. Our analysis identified approximately 62,000 expressed circRNAs in mCRC including approximately 32,000 expressed in patients and 48,000 expressed in cell lines, originating from approximately 9200 linear genes. Approximately 42,000 circRNAs (67%) were unannotated when compared with the circRNA consortium MiOncoCirc annotation. DE analysis identified 376 circRNAs deregulated in CRC progression from normal to primary, and metastasis. A higher proportion of DE circRNAs (82%) was downregulated in primary or metastasis, consistent with earlier reports of lower circRNA concentration in tumor due to rapid cell cycles. Among DE circRNAs were the downregulation of circRNAs that have been known to suppress tumor proliferation, migration, and invasion in mCRC such as circSMARCA5, circFBXW7, and circSETD3, and the upregulation of circFIRRE, a circRNA known to promote tumor progression in osteosarcoma. A significant portion (37%) of deregulated circRNAs also had their parental linear genes found to be deregulated, suggesting parental gene regulation contributed to circRNA regulation. Interestingly, a large portion of circRNAs (63%) were found to be deregulated while their parental linear gene expression was unchanged, suggesting alternative mechanisms of circRNA deregulation. Gene set enrichment analysis showed that linear genes co-deregulated with circRNAs were enriched in signatures of tumor progression including proliferation, epithelial mesenchymal transition, and metastasis, suggesting potential involvement of deregulated circRNAs in tumor progression. Through RNA sequencing of both patients and cell lines, we established a comprehensive compendium of circRNAs and identified those deregulated in mCRC. This provides a valuable resource for further experimental characterization of circRNAs function in mCRC progression. Citation Format: Sidi Zhao, Amy Ly, Jacqueline L. Mudd, Emily B. Rozycki, Ghofran Othoum, Jingqin Luo, Ha X. Dang, Ryan C. Fields, Christopher A. Maher. The landscape of circular RNAs in metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3803.

Functional heterogeneity of MCT1 and MCT4 in metabolic reprogramming affects osteosarcoma growth and metastasis

BackgroundOsteosarcoma is the most common primary malignant bone tumor in adolescents and children and prone to develop lung metastasis. Its prognosis has been virtually unimproved over the last few decades, especially in patients with metastases, who suffer from a dismal survival. Recently, increasing attention has been devoted to monocarboxylate transporters-related (MCTs) metabolic reprogramming. However, the role of MCT1 and MCT4 in osteosarcoma progression and the underlying mechanisms remain to be further elucidated.MethodsIn this study, we established MCT1 and/or MCT4 knockout cell lines by CRISPR/Cas9 genome editing technology. Then, we assessed glycolysis and oxidative phosphorylation capacities by measuring lactate flux and oxygen consumption. We also performed flowcytometry to test circulating tumor cells and PET/CT to evaluate glucose uptake.ResultsMCT1 was found to be involved in both glycolysis and oxidative respiration due to its ability to transport lactate in both directions. MCT1 inhibition significantly reduced circulating tumor cells and distant metastases partially by increasing oxidative stress. MCT4 was primarily related to glycolysis and responsible for lactate export when the concentration of extracellular lactate was high. MCT4 inhibition dramatically suppressed cell proliferation in vitro and impaired tumor growth with reduction of glucose uptake in vivo.ConclusionsOur results demonstrate the functional heterogeneity and redundancy of MCT1 and MCT4 in glucose metabolism and tumor progression in osteosarcoma. Thus, combined inhibition of MCT1 and MCT4 may be a promising therapeutic strategy for treating tumors expressing both transporters.

Deubiquitinase USP13 regulates glycolytic reprogramming and progression in osteosarcoma by stabilizing METTL3/m6A/ATG5 axis.

Reprogramming metabolism is a hallmark of cancer cells for rapid progression. However, the detailed functional role of deubiquitinating enzymes (DUBs) in tumor glycolytic reprogramming is still unknown and requires further investigation. USP13 was found to upregulate in osteosarcoma (OS) specimens and promote OS progression through regulating aerobic glycolysis. Interestingly, the m6A writer protein, METTL3, has been identified as a novel target of USP13. USP13 interacts with, deubiquitinates, and therefore stabilizes METTL3 at K488 by removing K48-linked ubiquitin chains. Since METTL3 is a well-known m6A writer and USP13 stabilizes METTL3, we further found that USP13 increased global m6A abundance in OS cells. The results of RNA sequencing and methylated RNA immunoprecipitation sequencing indicated METTL3 could bind to m6A-modified ATG5 mRNA, which is crucial for autophagosome formation, and inhibit ATG5 mRNA decay on an IGF2BP3 dependent manner, thereby promoting autophagy and the autophagy-associated malignancy of OS. Using a small-molecule inhibitor named Spautin-1 to pharmacologically inhibit USP13 induced METTL3 degradation and exhibited significant therapeutic efficacy both in vitro and in vivo. Collectively, our study results indicate that USP13 promotes glycolysis and tumor progression in OS by stabilizing METTL3, thereby stabilizing ATG5 mRNA and facilitating autophagy in OS. Our findings demonstrate the role of the USP13-METTL3-ATG5 cascade in OS progression and show that USP13 is a crucial DUB for the stabilization of METTL3 and a promising therapeutic target for treating OS.

ALKBH5 involves in osteosarcoma tumor progression by mediating Notch signaling

AbstractALKBH5 is the major demethylase of ribonuclease m6A and exerts these multiple biological functions in cancer. In this study, we mainly explore the use of ALKBH5 in the development and progression of osteosarcoma and elucidate the potential molecular mechanisms by which it functions. Relative to human osteoblast cell lines (NHOst), we detected ALKBH5 expression in osteosarcoma cell lines (HOS, U2OS, and MG‐63) by RT‐PCR and western blot. By overexpression and knockout of ALKBH5 gene, to observe the effect of ALKBH5 on apoptosis, invasion and epithelial‐mesenchymal transition (EMT) of osteosarcoma cells, as well as the effect of Notch signaling pathway. We found that ALKBH5 was significantly higher in osteosarcoma cell lines than in osteoblastic cell lines. In addition, ALKBH5 overexpression promoted the proliferation, migration, and invasion of osteosarcoma cells, and simultaneously activated Notch signaling pathway and up‐regulated E‐cadherin protein. Knockout of ALKBH5 inhibited these effects in osteosarcoma cells. It was also found that up‐regulation of ALKBH5 could promote the proliferation of osteosarcoma cells in vitro and in vivo. ALKBH5 can be involved in osteosarcoma development through Notch signaling pathway regulation. ALKBH5 is therefore expected to be a new target in the treatment of osteosarcoma.

MiR-659-3p inhibits osteosarcoma progression and metastasis by inhibiting cell proliferation and invasion via targeting SRPK1

ObjectiveOsteosarcoma is the most common primary bone cancer that affects mostly children and young adults. Despite the advances in osteosarcoma treatment, the long-term survival rate of metastatic patients has not significantly improved in the past few decades, thus demonstrating the need for novel therapeutic targets or methods to improve metastatic osteosarcoma treatment. In this study we aimed to elucidate the role of miR-659-3p and SRPK1 in osteosarcoma.MethodsWe evaluated miR-659-3p and SRPK1 function in osteosarcoma cell proliferation, migration, and cell cycle progression in vitro by using gain- and loss-of-function strategies. The effect of miR-659-3p in tumor progression and metastasis was determined by in vivo mouse model.ResultsWe revealed that expression of miR-659-3p was significantly downregulated in osteosarcoma compared with normal bone cells and was inversely correlated with serine-arginine protein kinase 1 (SRPK1) expression. We proved that miR-659-3p targets 3’ UTR of SRPK1 and negatively regulates SRPK1 expression in osteosarcoma cells via luciferase assay. In vitro studies revealed that gain of miR-659-3p function inhibited osteosarcoma cells growth, migration, and invasion by down-regulating SRPK1 expression. Inversely, inhibiting miR-659-3p in osteosarcoma cells promoted cell growth, migration, and invasion. Cell cycle profile analysis revealed that miR-659-3p inhibited osteosarcoma cells’ G1/G0 phase exit by down-regulating SRPK1 expression. By using an in vivo mouse model, we demonstrated that miR-659-3p inhibits osteosarcoma tumor progression and lung metastasis by inhibiting SRPK1 expression and potentially downstream cell proliferation, and epithelial-to-mesenchymal transition genes.ConclusionsThis study demonstrated that miR-659-3p is a potential therapeutic method and SRPK1 is a potential therapeutic target for osteosarcoma treatment.

MerTK-mediated efferocytosis promotes immune tolerance and tumor progression in osteosarcoma through enhancing M2 polarization and PD-L1 expression

ABSTRACT The poor progress of immunotherapy on osteosarcoma patients requires deeper delineation of immune tolerance mechanisms in the osteosarcoma microenvironment and a new therapeutic strategy. Clearance of apoptotic cells by phagocytes, a process termed “efferocytosis,” is ubiquitous in tumors and mediates the suppression of innate immune inflammatory response. Considering the massive infiltrated macrophages in osteosarcoma, efferocytosis probably serves as a potential target, but is rarely studied in osteosarcoma. Here, we verified M2 polarization and PD-L1 expression of macrophages following efferocytosis. Pharmacological inhibition and genetic knockdown were used to explore the underlying pathway. Moreover, tumor progression and immune landscape were evaluated following inhibition of efferocytosis in osteosarcoma model. Our study indicated that efferocytosis promoted PD-L1 expression and M2 polarization of macrophages. Ëfferocytosis was mediated by MerTK receptor in osteosarcoma and regulated the phenotypes of macrophages through the p38/STAT3 pathway. By establishing the murine osteosarcoma model, we emphasized that inhibition of MerTK suppressed tumor growth and enhanced the T cell cytotoxic function by increasing the infiltration of CD8+ T cells and decreasing their exhaustion. Our findings demonstrate that MerTK-mediated efferocytosis promotes osteosarcoma progression by enhancing M2 polarization of macrophages and PD-L1-induced immune tolerance, which were regulated through the p38/STAT3 pathway.

Exosomal Long Non-Coding RNA ANCR Mediates Drug Resistance in Osteosarcoma.

Osteosarcoma (OS) is rare cancer with bimodal age distribution with peaks observed in children and young adults. Typically, OS is treated with pre-surgery neoadjuvant therapy, surgical excision, and post-surgery chemotherapy. However, the efficacy of treatment on disease prognosis and objective response is not currently optimal, often resulting in drug resistance; in turn, highlighting the need to understand mechanisms driving resistance to therapy in OS patients. Using Doxycycline (Dox)-sensitive and resistant variants of OS cells lines KHOS and U2OS, we found that the resistant variants KHOS-DR and U2OS-DR have significantly higher in vitro proliferation. Treating the Dox-sensitive KHOS/U2OS cells with exosomes isolated from KHOS-DR/U2OS-DR made them resistant to treatment with Dox in vitro and in vivo and enhanced tumor growth and progression, while decreasing overall survival. Expression of the long non-coding RNA (lncRNA) ANCR was significantly higher in the KHOS-DR and U2OS-DR variants. SiRNA-mediated knockdown of ANCR decreased in vitro proliferation, while increasing sensitivity to Dox treatment in the KHOS-DR/U2OS-DR cells. Expression of the exosomal lncRNA ANCR was critical for drug resistance and OS tumor progression in xenografts and was correlated to resistance to Adriamycin and overall survival is patients with OS. These results establish lncRNA ANCR as a critical mediator of resistance to therapy in OS patients, highlighting it as a potential therapeutic target in OS patients.

MiR-183-5p Promotes Tumor Progression of Osteosarcoma and Predicts Poor Prognosis in Patients.

BackgroundAlthough miRNA-183-5p plays a critical role in many cancer types, including gastric cancer, hepatocellular carcinoma, prostate cancer, renal cell cancer and breast cancer, its role in osteosarcoma remains unclear.MethodsExpression levels of miR-183-5p were detected in osteosarcoma tissues and cell lines using qRT-PCR. The effect of miR-183-5p on the survival and recurrence of osteosarcoma patients was analyzed in a cohort of 80 patients using Kaplan–Meier curves and Cox regression analysis. Effects of miR-183-5p on cell proliferation, migration and invasion abilities were evaluated using CCK-8, crystal violet and transwell assays.ResultsThe expression of miR-183-5p was found to be upregulated in human osteosarcoma tissues and cell lines. Moreover, miR-183-5p expression was observed to be closely associated with tumor size, TNM stage and lung metastasis. Notably, high expression of miR-183-5p was shown to be able to predict unfavorable clinical prognosis in osteosarcoma patients. Additionally, whilst overexpression of miR-183-5p was observed to significantly promote the proliferation, migration and invasion of osteosarcoma cells; an inhibitory effect was observed with knockdown of miR-183-5p.ConclusionThis study demonstrated that miR-183-5p acts as an oncogene and plays a critical role in the regulation of osteosarcoma tumor progression, and our results suggest a novel potential prognostic and therapeutic value of miR-183-5p in osteosarcoma.

MicroRNA-761 suppresses tumor progression in osteosarcoma via negatively regulating ALDH1B1

AimsOur previous study has demonstrated that high expression of ALDH1B1 promoted osteosarcoma tumor progression and was correlated with unfavorable prognosis in osteosarcoma patients. In the current study, we investigated the underlying mechanism and regulation of ALDH1B1 in osteosarcoma. Materials and methodsqRT-PCR assay was applied to detect miR-761 expression. CCK-8, colony formation and EdU assays were conducted to explore the functional role of miR-761/ALDH1B1 axis in osteosarcoma. Bioinformatics analysis and luciferase reporter assay was utilized to assess the regulation between miR-761 and ALDH1B1. Mechanism experiments were implemented to investigate the underlying molecular mechanism of miR-761/ALDH1B1 axis. Key findingsALDH1B1 was negatively regulated by microRNA-761 (miR-761). Functionally, miR-761 suppressed cell growth, migration, and invasion in osteosarcoma via targeting ALDH1B1 in vitro. Xenograft tumor model demonstrated that miR-761 inhibited osteosarcoma tumor development in vivo through regulating ALDH1B1. Consistently, we showed that miR-761 expression was decreased in osteosarcoma patients and low expression of miR-761 was correlated with worse prognosis in osteosarcoma patients. Mechanistically, we revealed that high expression of ALDH1B1 was significantly associated with enhanced TGF-β signaling, epithelial-mesenchymal transition (EMT), and cell adhesion. Furthermore, miR-761 regulated TGF-β and EMT/cell adhesion in osteosarcoma via targeting ALDH1B1. SignificanceTaken together, our findings suggest that the oncogenic ALDH1B1 is regulated by miR-761 during osteosarcoma development and progression, which might provide a novel prognostic biomarker and therapeutic strategy for osteosarcoma treatment.

Bone marrow mesenchymal stem cell-derived exosomal miR-206 inhibits osteosarcoma progression by targeting TRA2B.

Osteosarcoma is the most common primary malignant bone tumor in young people. Recently, extracellular vesicles, especially exosomes, have been reported to play an increasingly important role in the development of many types of tumors. In this research, we found that overexpression of transformer 2β (TRA2B) was associated with tumor progression in osteosarcoma, and TRA2B was the target gene of miR-206, which was downregulated in osteosarcoma tissues. Furthermore, we observed that bone marrow mesenchymal stem cell (BMSC)-derived exosomes could carry and transport miR-206 to osteosarcoma cells. Both in vitro and in vivo results showed that BMSC-derived exosomal miR-206 could inhibit the proliferation, migration and invasion of osteosarcoma cells and induce their apoptosis. Taken together, our study demonstrates that BMSC-derived exosomal miR-206 can be transferred into osteosarcoma cells and inhibit tumor progression by targeting TRA2B, which provides new insight into the molecular mechanism of osteosarcoma and highlights the potential of miR-206 and TRA2B as new therapeutic targets.

Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in osteosarcoma.

Osteosarcoma is the most common primary malignancy of bones and frequently affects young children and adolescents. There are several challenges associated with treating osteosarcoma owing to the aggressiveness of the disease, as well as the risk of chemoresistance. Numerous studies are being performed with the aim of identifying improved prognostic and therapeutic markers for this malignancy. Maternal embryonic leucine zipper kinase (MELK) is an oncogene that has been studied in several types of cancer in recent years. In the present study, the expression of MELK in osteosarcoma and normal tissue samples was examined, and the effects of MELK expression on osteosarcoma cellular proliferation, metastasis, the cell cycle and apoptosis were demonstrated using CCK-8, wound healing, migration and invasion and apoptosis assays. The role of MELK in cancer progression in osteosarcoma was determined, revealing the association between MELK expression and prognosis of osteosarcoma. It was demonstrated that knockdown of MELK resulted in reduced proliferation, migration and invasion in vitro along with potentiation of apoptosis and cell cycle arrest. Furthermore, the effect of the targeted MELK inhibitor, OTSSP167, on tumor progression of osteosarcoma in vitro and in vivo was assessed. Mechanistically, it was demonstrated that MELK promoted osteosarcoma proliferation and metastasis by regulating PCNA and MMP9 expression via the PI3K/Akt/mTOR signaling pathway. Thus, the present study revealed the oncogenic role played by MELK, and established MELK as a valuable prognostic and therapeutic marker in osteosarcoma.

Caprin1 targeted by circular circ_0000885 in the tumor progression of osteosarcoma.

Recent studies have revealed that circular RNAs (circRNAs) participate in the progression and development of many human diseases. Therefore, the purpose of this study was to uncover the role of circ_0000885 in the development of osteosarcoma and to explore the possible underlying mechanism. Circ_0000885 expression in osteosarcoma tissues and cells was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The knockdown of circ_0000885 was conducted in osteosarcoma cells. Subsequently, its function in the progression of osteosarcoma was determined by cell proliferation assay, colony formation assay, and cell cycle assay, respectively. Furthermore, the underlying mechanism was explored by RT-qPCR and Western blot. Circ_0000885 was highly expressed in osteosarcoma tissues than that of adjacent tissues. Cell growth was significantly suppressed, and the distribution of cell cycle was regulated after circ_0000885 knock-down in vitro. Besides, both the mRNA and protein expressions of Caprin1 were significantly downregulated via silence of circ_0000885. Furthermore, Caprin1 was positively correlated with circ_0000885 in osteosarcoma tissues. Circ_0000885 could enhance osteosarcoma cell proliferation and regulate cell cycle by upregulating Caprin1, which might contribute to the therapy for osteosarcoma.

KIF18B promotes tumor progression in osteosarcoma by activating β-catenin.

Objective: Osteosarcoma is a common primary highly malignant bone tumor. Kinesin family member 18B (KIF18B) has been identified as a potential oncogene involved in the development and metastasis of several cancer types. While KIF18B overexpression in osteosarcoma tissue is clearly detected, its specific function in the disease process remains to be established.Methods: KIF18B expression was assessed in osteosarcoma tissues and cells. We additionally evaluated the effects of KIF18B on proliferation, migration, and invasion of osteosarcoma cells, both in vitro and in vivo.Results: Our results showed overexpression of KIF18B in osteosarcoma tissues and cells. Knockdown of KIF18B induced G1/S phase arrest and significantly inhibited proliferation, migration, and invasion of osteosarcoma cells, both in vitro and in vivo. KIF18B regulated β-catenin expression at the transcriptional level by controlling nuclear aggregation of ATF2 and at the post-transcriptional level by interacting with the adenomatous polyposis coli (APC) tumor suppressor gene in osteosarcoma cells.Conclusions: KIF18B plays a carcinogenic role in osteosarcoma by regulating expression of β-catenin transcriptionally via decreasing nuclear aggregation of ATF2 or post-transcriptionally through interactions with APC. Our collective findings support the potential utility of KIF18B as a novel prognostic biomarker for osteosarcoma.

MiR-384 Inhibits Malignant Biological Behavior Such as Proliferation and Invasion of Osteosarcoma by Regulating IGFBP3.

Osteosarcoma is the most common primary malignant bone tumor in the clinic. It is more common in children and adolescents. It has high malignancy, early metastasis rate, rapid disease progression, and high mortality. Although past years have witnessed the great improvement in the treatments of osteosarcoma, there remains a long way to go. MicroRNAs affect the malignant biological behaviors such as tumor proliferation and metastasis by regulating their target genes. In this study, we investigated the role and mechanism of miR-384 in osteosarcoma. Quantitative real-time polymerase chain reaction assay was performed to detect the expression of miR-384 and insulin-like growth factor binding protein 3 in osteosarcoma tissues and cell lines and established its correlation with osteosarcoma tumor progression and metastasis. To probe whether miR-384 played a tumor suppression role in osteosarcoma, we carried out gain-of-function and loss-of-function assays. Cell Counting Kit-8, cell colony formation, and transwell assays were carried out to determine the cells proliferation and invasion, respectively. Western blot was used to detect the changes of epithelial–mesenchymal transition marker proteins and insulin-like growth factor binding protein 3. MiR-384 was downregulated in osteosarcoma tissues and cell lines. MiR-384 was overexpressed in G292 cells transfected with miR-384 mimics and knocked down in Saos-2 cells with small hairpin RNA targeting miR-384. Ectopic expression of miR-384 inhibited osteosarcoma cell proliferation, colony formation, and invasion. E-cadherin was brought to a decrease whereas N-cadherin and Snail to an increase under the silent expression of miR-384, while overexpression of miR-384 led to an opposite result. MiR-384 could regulate insulin-like growth factor binding protein 3 expression in osteosarcoma. Quantitative polymerase chain reaction and Western blotting results validated that miR-384 knockdown downgrades both messenger RNA and protein levels of insulin-like growth factor binding protein 3 in G292 cells, while miR-384 upregulation exerted an opposite effect in Saos-2 cells. Insulin-like growth factor binding protein 3 was upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with normal ones. Through the bioinformatics database found that the upstream transcriptional regulator of insulin-like growth factor binding protein 3 is MECP2. So miR-384 can directly inhibit MECP2 and then promote the expression of insulin-like growth factor binding protein 3. These results suggested that miR-384 might be a potential therapeutic targets and biomarker in osteosarcoma.

LncRNA PCAT6 promotes tumor progression in osteosarcoma via activation of TGF-β pathway by sponging miR-185-5p

Long noncoding RNAs (lncRNAs) play crucial roles in tumor development of osteosarcoma (OS). LncRNA PCAT6 was involved in the progression of multiple human cancers. However, the biological function of PCAT6 in OS remains largely unknown. We found that PCAT6 was elevated in OS tissues relative to that in their adjacent normal tissues. The upregulation of PCAT6 was positively associated with metastasis status and advanced stages and predicted poor overall and progression-free survivals in patients with OS. Functionally, silencing PCAT6 inhibited the proliferation, migration and invasion abilities of OS cells. Mechanistically, PCAT6, acting as a competitive endogenous RNA, upregulated expression of TGFBR1 and TGFBR2 to activate TGF-β pathway via sponging miR-185–5p. This study uncovers a novel underlying molecular mechanism of PCAT6-miR-185-5p-TGFBR1/2-TGF-β signaling axis in promoting tumor progression in OS, which indicates that PCAT6 may serve as a promising prognostic factor and therapeutic target again OS.

LINC00324 accelerates the proliferation and migration of osteosarcoma through regulating WDR66.

Osteosarcoma (OS) is a type of malignancy featured with high morbidity and easy metastasis. Although past years have witnessed the great improvement in the treatments of OS, there remains a long way to go. Therefore, further research on the underlying molecular mechanism of OS progression is in imminent need. Long noncoding RNAs (lncRNA) are recognized as a cluster of transcripts over 200 bases. Increasing studies have unveiled their significant regulatory roles in cancers, including in osteosarcoma. Long intergenic non-protein coding RNA 324 (LINC00324) is a newly identified lncRNA exerting oncogenic functions in several cancers, but its role in OS is yet to be uncovered. Therefore, the present study planned to explore the role of LINC00324 in osteosarcoma. We first validated the upregulation of LINC00324 in OS tissues and cell lines and established its correlation with OS tumor progression and metastasis. Importantly, the prognostic significance of LINC00324 was identified in patients with OS. Gain- and loss-of-function assays revealed that LINC00324 accelerated cell proliferation and migration in OS. Mechanistically, we revealed that LINC00324 stabilized WD repeat-containing protein 66 (WDR66) messenger RNA through interacting with Hu antigen R. Rescue assays verified that WDR66 was required for the regulation of LINC00324 in promoting proliferation and migration of OS cells. In conclusion, the present study proved that LINC00324 accelerated the proliferation and migration of osteosarcoma cells through regulating WDR66, providing a new prognostic target for osteosarcoma.

Carbonic anhydrase IX enhances tumor cell proliferation and tumor progression in osteosarcoma.

PurposeWe investigated the effect of carbonic anhydrase IX (CA IX) inhibitor under hypoxia and normoxia in SaOS2 human osteosarcoma cell line. We also evaluated the expression of CA IX in 27 patients diagnosed with osteosarcoma.Materials and methodsCA IX expression in SaOS2 cells cultured under different oxygen tensions was analyzed by Western blotting. To evaluate the effect of CA IX inhibitor, MTS cell viability assay was performed after cells were treated with various concentrations of doxorubicin with or without a CA IX inhibitor. Finally, CA IX expression in patient-derived osteosarcoma samples was evaluated by immunohistochemistry.ResultsTreatment with CA IX inhibitor significantly suppressed cell proliferation and migration under hypoxic conditions. CA IX expression was observed in 81% of 27 patients. The 5-year survival rates in patients with high and low stain scores were 43.8% and 81.8%, respectively.ConclusionCA IX inhibitors have the potential to suppress cell proliferation, migration, and chemoresistance.

Super enhancer inhibitors suppress MYC driven transcriptional amplification and tumor progression in osteosarcoma

Osteosarcoma is the most common primary bone sarcoma that mostly occurs in young adults. The causes of osteosarcoma are heterogeneous and still not fully understood. Identification of novel, important oncogenic factors in osteosarcoma and development of better, effective therapeutic approaches are in urgent need for better treatment of osteosarcoma patients. In this study, we uncovered that the oncogene MYC is significantly upregulated in metastastic osteosarcoma samples. In addition, high MYC expression is associated with poor survival of osteosarcoma patients. Analysis of MYC targets in osteosarcoma revealed that most of the osteosarcoma super enhancer genes are bound by MYC. Treatment of osteosarcoma cells with super enhancer inhibitors THZ1 and JQ1 effectively suppresses the proliferation, migration, and invasion of osteosarcoma cells. Mechanistically, THZ1 treatment suppresses a large group of super enhancer containing MYC target genes including CDK6 and TGFB2. These findings revealed that the MYC-driven super enhancer signaling is crucial for the osteosarcoma tumorigenesis and targeting the MYC/super enhancer axis represents as a promising therapeutic strategy for treatment of osteosarcoma patients.

The long noncoding RNA cancer susceptibility candidate 2 inhibits tumor progression in osteosarcoma.

Long noncoding RNA (lncRNA) has been identified to serve a critical role in the development of various types of cancer. Cancer susceptibility candidate 2 (CASC2) is a cancer‑associated lncRNA. However, whether CASC2 regulates osteosarcoma progression remains unclear. Reverse transcription‑quantitative polymerase chain reaction, western blot, invasion and migration assays were used to evaluate the role of CASC2 in osteosarcoma. The present study reported that CASC2 may inhibit osteosarcoma development. Osteosarcoma tissues demonstrated reduced CASC2 expression compared with normal adjacent tissues. In addition, CASC2 transduction may decrease proliferation, migration and invasion of osteosarcoma cell lines whereas knockdown of CASC2 displayed opposing effects. Patients with low CASC2 levels were predicted to have a poor survival. Invivo implantation studies using pcDNA‑CASC2 or short interfering‑CASC2 exhibited decreased or increased tumor weight, respectively. These results suggested that CASC2 may serve as a potential tumor suppressor lncRNA in osteosarcoma and may provide potential insight into targeted intervention.