Promotes Osteosarcoma Progression Research Articles

VCP enhances autophagy-related osteosarcoma progression by recruiting USP2 to inhibit ubiquitination and degradation of FASN

Osteosarcoma (OS) is a highly aggressive malignant tumor with a high rate of disability and mortality rates, and dysregulated autophagy is a crucial factor in cancer. However, the molecular mechanisms that regulate autophagy in OS remain unclear. This study aimed to explore key molecules that affect autophagy in OS and their regulatory mechanisms. We found that fatty acid synthase (FASN) was significantly increased in activated autophagy models of OS and promoted OS proliferation in an autophagy-dependent manner, as detected by LC3 double-labeled fluorescence confocal microscopy, western blotting, transmission electron microscopy (TEM), and cell functional experiments. Furthermore, co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), ubiquitination modification, molecular docking, and protein truncation methods were used to identify FASN-interacting proteins and analyze their effects on OS. Valosin-containing protein (VCP) enhanced the FASN stability by recruiting ubiquitin specific peptidase-2 (USP2) to remove the K48-linked ubiquitin chains from FASN; domain 2 of VCP and the amino acid sequence () of USP2 were critical for their interactions. Gain- and loss-of-function experiments showed that the inhibition of FASN or USP2 attenuated the stimulatory effect of VCP overexpression on autophagy and the malignant phenotypes of OS cells in vitro and in vivo. Notably, micro-CT indicated that VCP induced severe bone destruction in nude mice, which was abrogated by FASN or USP2 downregulation. In summary, VCP recruits USP2 to stabilize FASN by deubiquitylation, thereby activating autophagy and promoting OS progression. The identification of the VCP/USP2/FASN axis, which mediates autophagy regulation, provides important insights into the underlying mechanisms of OS and offers potential diagnostic and therapeutic strategies for patients with OS.

LncRNA POU3F3 promotes osteosarcoma progression through GPX4-modulated ferroptosis by interaction with IGF2BP2 to facilitate NRF2 mRNA stability

lncRNA POU3F3 promotes osteosarcoma progression through GPX4-modulated ferroptosis by interaction with IGF2BP2 to facilitate NRF2 mRNA stability

Small Nuclear Ribonucleoprotein Polypeptides B and B1 Promote Osteosarcoma Progression via Activating the Ataxia-Telangiectasia Mutated Signaling Pathway through Ribonucleotide Reductase Subunit M2

Osteosarcoma is a malignant bone tumor characterized by high metastatic potential and recurrence rates post-therapy. The small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB), a core component of spliceosome, have been reported to exhibit upregulation across several cancer types. However, the precise role of SNRPB in osteosarcoma progression remains poorly elucidated. Herein, we explored SNRPB expression in human osteosarcoma tissues and normal bone tissues by immunohistochemical staining (IHC) staining, revealing a notable upregulation of SNRPB in osteosarcoma, correlating with diminished survival rates. Moreover, the in vitro loss-of-function experiments showed that SNRPB knockdown significantly suppressed the osteosarcoma cell proliferation and migration, as well as tubule formation of HUVECs, while enhancing osteosarcoma cell apoptosis. Mechanistically, we revealed that SNRPB promoted the transcription of ribonucleotide reductase subunit M2 (RRM2) via E2F transcription factor 1 (E2F1). Further rescue experiments indicated that RRM2 was required for SNRPB-induced malignant behaviors in osteosarcoma. Additionally, we confirmed that the function of SNRPB in osteosarcoma cell growth and apoptosis was associated with ATM signaling pathway activation. In conclusion, our findings provide initial insights into the underlying mechanisms governing SNRPB-induced osteosarcoma progression and proposed for SNRPB as a novel therapeutic target in osteosarcoma management.

LncRNA-PVT1 Inhibits Ferroptosis through Activating STAT3/GPX4 Axis to Promote Osteosarcoma Progression.

Osteosarcoma (OS) is a primary malignant bone tumor in the pediatric and adolescent populations. Long non-coding RNAs (LncRNAs), such as plasma-cytoma variant translocation 1 (PVT1), have emerged as significant regulators of OS metastasis. Recent studies have indicated that activation of signal transducer and activator of transcription 3 (STAT3) signaling, which might be controlled by PVT1, inhibits ferroptosis to promote the malignant progression of cancer. Therefore, the present study aimed to determine the role of PVT1 in OS pathogenesis and investigate whether PVT1 affects OS progression by regulating STAT3/GPX4 pathway-mediated ferroptosis. The human OS cell line MG63 were transfected with sh-PVT1 plasmid to inhibit PVT1 expression, with or without co-transfection with a STAT3 overexpression plasmid. The expression of PVT1 was determined by real-time quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, invasion, and apoptosis of MG63 cells were determined using the cell counting kit-8 (CCK8), Transwell assay, and flow cytometry. The levels of malondialdehyde (MDA), Fe2+, and glutathione (GSH) were determined by ELISA kits, whereas reactive oxygen species (ROS) level was determined by immunofluorescence. The protein expression levels of STAT3, p-STAT3, and glutathione peroxidase 4 (GPX4) were detected by western blot (WB). PVT1 expression was significantly increased in MG63 cells. When knocking down PVT1 with sh-PVT1 plasmid, the proliferation, migration, and invasion of MG63 cells were markedly inhibited, while the rate of apoptosis was upregulated. Further investigation revealed that MG63 cells with PVT1 knockdown exhibited elevated levels of MDA, Fe2+, and ROS. In addition, the inhibition of PVT1 expression resulted in decreased levels of GSH and inhibited expression of p-STAT3 and GPX4. When sh-PVT1 was co-transfected with STAT3 overexpression plasmid in MG63 cells, the increased levels of MDA, Fe2+, and ROS were downregulated, and the decreased expressions of GSH, p-STAT3, and GPX4 were upregulated. PVT1 promotes OS metastasis by activating the STAT3/GPX4 pathway to inhibit ferroptosis. Targeting PVT1 might be a novel therapeutic strategy for OS treatment.

USP22 as a key regulator of glycolysis pathway in osteosarcoma: insights from bioinformatics and experimental approaches.

Osteosarcoma is the most common primary malignant bone tumor, but its pathogenesis remains unclear. Ubiquitin-specific processing peptidase 22 (USP22) is reported to be highly expressed and associated with tumor malignancy and prognosis in cancers. However, the role and mechanism of USP22 in osteosarcoma is not fully understood. This study aims to investigate the function and potential mechanism of USP22 in osteosarcoma using bioinformatics analysis combined with experimental validation. We first integrated transcriptomic datasets and clinical information of osteosarcoma from GEO and TCGA databases to assess the expression and prognostic value of USP22 in osteosarcoma. Then, differential expression analysis and weighted gene co-expression network analysis (WGCNA) were conducted to identify USP22-related co-expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the biological functions and signaling pathways of USP22 co-expressed genes. To validate the accuracy of bioinformatics analyses, we downregulated USP22 expression in osteosarcoma cell line Sao-2 using siRNA and assessed its effect on cell proliferation, migration, invasion, apoptosis, and regulation of key signaling pathways. We found that USP22 was highly expressed in osteosarcoma tissues and correlated with poor prognosis in osteosarcoma patients. USP22 also showed potential as a diagnostic marker for osteosarcoma. In addition, 344 USP22-related co-expressed genes were identified, mainly involved in signaling pathways such as glycolysis, oxidative phosphorylation, spliceosome, thermogenesis, and cell cycle. The in vitro experiments confirmed the accuracy and reliability of bioinformatics analyses. We found that downregulation of USP22 could inhibit Sao-2 cell proliferation, migration, invasion, and induce apoptosis. Furthermore, downregulation of USP22 significantly reduced aerobic glycolysis levels in Sao-2 cells and inhibited the expression of key enzymes and transporters in aerobic glycolysis pathways such as HK2, PKM2, and GLUT1. USP22 plays a critical role in the occurrence, development, and prognosis of osteosarcoma. USP22 could influence Sao-2 cell proliferation, apoptosis, migration, and invasion by regulating the glycolysis pathway, thereby promoting osteosarcoma progression. Therefore, USP22 may be a potential therapeutic target for the treatment of osteosarcoma.

High expression of SRSF1 facilitates osteosarcoma progression and unveils its potential mechanisms

BackgroundSRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS.MethodsSRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1.ResultsSRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS.ConclusionsOur results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.

Upregulation of circ_0076684 in osteosarcoma facilitates malignant processes by mediating miRNAs/CUX1

Circular RNAs (circRNAs) are a newly appreciated type of endogenous noncoding RNAs that play vital roles in the development of various human cancers, including osteosarcoma (OS). In this study, we investigated three circRNAs (circ_0076684, circ_0003563, circ_0076691) from the RUNX Family Transcription Factor 2 (RUNX2) gene locus in OS. We found that the expression of circ_0076684, circ_0003563, circ_0076691, and RUNX2 mRNA is upregulated in OS, which is a consequence of CBX4-mediated transcriptional activation. Among these three RUNX2-circRNAs, only circ_0076684 is significantly associated with the clinical features and prognosis of OS patients. Functional experiments indicate that circ_0076684 promotes OS progression in vitro and in vivo. Circ_0076684 acts as a sponge for miR-370-3p, miR-140-3p, and miR-193a-5p, raising Cut Like Homeobox 1 (CUX1) expression by sponging these three miRNAs. Furthermore, we presented that circ_0076684 facilitates OS progression via CUX1. In conclusion, this study found that the expression of three circRNAs and RUNX2 mRNA from the RUNX2 gene locus is significantly upregulated in OS, as a result of CBX4-mediated transcriptional activation. Circ_0076684 raises CUX1 expression by sponging miR-370-3p, miR-140-3p, and miR-193a-5p, and facilitates OS progression via CUX1.

POLE2 promotes osteosarcoma progression by enhancing the stability of CD44.

Osteosarcoma (OS) is the most prevalent primary malignancy of bone in children and adolescents. It is extremely urgent to develop a new therapy for OS. In this study, the GSE14359 chip from the GEO database was used to screen differentially expressed genes in OS. DNA polymerase epsilon 2 (POLE2) was confirmed to overexpress in OS tissues and cell lines by immunohistochemical staining, qPCR and Western blot. Knockdown of POLE2 inhibited the proliferation and migration of OS cells in vitro, as well as the growth of tumors in vivo, while the apoptosis rate was increased. Bioinformatics analysis revealed that CD44 and Rac signaling pathway were the downstream molecule and pathway of POLE2, which were inhibited by knockdown of POLE2. POLE2 reduced the ubiquitination degradation of CD44 by acting on MDM2. Moreover, knockdown of CD44 inhibited the tumor-promoting effects of POLE2 overexpression on OS cells. In conclusion, POLE2 augmented the expression of CD44 via inhibiting MDM2-mediated ubiquitination, and then activated Rac signaling pathway to influence the progression of OS, indicating that POLE2/CD44 might be potential targets for OS treatment.

Therapeutic potential of tyrosine-protein kinase MET in osteosarcoma.

Osteosarcoma, the most prevalent primary bone tumor in children and young adults, can often be successfully treated with standard chemotherapy and surgery when diagnosed at an early stage. However, patients presenting with metastases face significant challenges in achieving a cure. Despite advancements in classical therapies over the past few decades, clinical outcomes for osteosarcoma have not substantially improved. Recently, there has been increased understanding of the biology of osteosarcoma, leading to the identification of new therapeutic targets. One such target is MET, a tyrosine kinase receptor for Hepatocyte Growth Factor (HGF) encoded by the MET gene. In vitro and in vivo studies have demonstrated that the HGF/MET pathway plays a crucial role in cancer growth, invasion, metastasis, and drug resistance across various cancers. Clinical trials targeting this pathway are already underway for lung cancer and hepatocellular carcinoma. Moreover, MET has also been implicated in promoting osteosarcoma progression. This review summarizes 3decades' worth of research on MET's involvement in osteosarcoma and further explores its potential as a therapeutic target for patients with this disease.

USP3 promotes osteosarcoma progression via deubiquitinating EPHA2 and activating the PI3K/AKT signaling pathway

Ubiquitin-specific protease 3 (USP3) plays an important role in the progression of various tumors. However, the role of USP3 in osteosarcoma (OS) remains poorly understood. The aim of this study was to explore the biological function of USP3 in OS and the underlying molecular mechanism. We found that OS had higher USP3 expression compared with that of normal bone tissue, and high expression of USP3 was associated with poor prognosis in patients with OS. Overexpression of USP3 significantly increased OS cell proliferation, migration, and invasion. Mechanistically, USP3 led to the activation of the PI3K/AKT signaling pathway in OS by binding to EPHA2 and then reducing its protein degradation. Notably, the truncation mutant USP3-F2 (159–520) interacted with EPHA2, and amino acid 203 was found to play an important role in this process. And knockdown of EPHA2 expression reversed the pro-tumour effects of USP3-upregulating. Thus, our study indicates the USP3/EPHA2 axis may be a novel potential target for OS treatment.

Retracted: Circular RNA circFAT1(e2) Promotes Osteosarcoma Progression and Metastasis by Sponging miR-181b and Regulating HK2 Expression.

[This retracts the article DOI: 10.1155/2020/3589871.].

Retraction: The long non-coding RNA-ROR promotes osteosarcoma progression by targeting miR-206.

Dan Fei, Guoqing Sui, Yang Lu, Long Tan, Zhao Dongxu, Kewei Zhang. The long non-coding RNA-ROR promotes osteosarcoma progression by targeting miR-206. J Cell Mol Med. 2019; 23: 1865-1872. https://doi.org/10.1111/jcmm.14087. The above article, published online on 19 December 2018 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, Stefan Constantinescu, The Foundation for Cellular and Molecular Medicine and John Wiley and Sons Ltd. The retraction has been agreed following an investigation into concerns raised by a third party, which revealed inappropriate duplications of images between this and other articles that were previously published in a different scientific context. Thus, the editors consider the conclusions of this manuscript substantially compromised. Although the authors requested a retraction, when asked to approve the retraction wording, they remained unresponsive.

LINC00629, a HOXB4-downregulated long noncoding RNA, inhibits glycolysis and ovarian cancer progression by destabilizing c-Myc.

Ovarian cancer (OC) cells typically reprogram their metabolism to promote rapid proliferation. However, the role of long noncoding RNAs (lncRNAs) in the metabolic reprogramming of ovarian cancer, especially in glucose metabolic reprogramming, remains largely unknown. LINC00629 has been reported in our previous study to promote osteosarcoma progression. Upregulated LINC00629 was found to enhance the growth-suppressive effect of apigenin on oral squamous cell carcinoma. However, the precise function of LINC00629 in ovarian cancer development remains poorly understood. In this study, we found that LINC00629 was significantly downregulated in OC tissues and that low LINC00629 expression was associated with poor survival. Inhibition of LINC00629 was required for increased glycolysis activity and cell proliferation in ovarian cancer. Invivo, overexpression of LINC00629 dramatically inhibited tumor growth and lung metastasis. Mechanistically, LINC00629 interacted with and destabilized c-Myc, leading to its ubiquitination and proteasome degradation, further resulting in increased expression of downstream glycolysis-related genes and glucose metabolic reprogramming in OC. Interestingly, HOXB4 bound to the LINC00629 promoter and inhibited its transcription, indicating that LINC00629 is a transcriptional target of HOXB4. Collectively, these findings establish a direct role for LINC00629 in suppressing glucose metabolism, and HOXB4/LINC00629/c-Myc might serve as a potential biomarker and an effective therapeutic strategy for OC cancer treatment.

Lnc-CLSTN2-1:1 Promotes Osteosarcoma Progression by Disrupting Redox Balance through PI3K/AKT Signaling Pathway.

Objective: Most patients with osteosarcoma (OS) have an extremely poor prognosis. The primary purpose of this investigation was to explore the biological effect of Lnc-CLSTN2-1:1 on OS and the potential processes involved. Materials and procedures: We selected differentially overexpressed Lnc-CLSTN2-1:1 from our laboratory's existing RNA sequence analysis data (fibroblast osteoblast (hFOB 1.19) and three osteosarcoma cell lines (HOS, MG63, and U2OS) as the research object. Next, we detected Lnc-CLSTN2-1:1 in the osteosarcoma HOS cell line and fibroblast cells using qRT-PCR. We evaluated cell proliferation ability using EdU incorporation test, CCK-8 test, and cell clone formation; cell invasion and migration were assessed using the Transwell test, while flow cytometry examined cell cycle, apoptosis, and reactive oxygen species (ROS); Subsequently, the activity changes of selenase (GPx) glutathione peroxidase and (TrxR) thioredoxin reductase were detected. In addition, changes in related proteins were analyzed through Western blotting. Results: The expression of Lnc-CLSTN2-1:1 in osteosarcoma cells was significantly increased. The proliferation, invasion, and migration of osteosarcoma cells were significantly inhibited by knockdown of the expression of Lnc-CLSTN2-1:1, and the cell cycle-related signaling pathway PI3K/AKT/GSK-3β/cycinD1 was also inhibited. However, insulin-like growth factor-1 (igf-1) could reverse this process. In addition, we examined the activity of two selenophenases (TrxR and GPx) and the changes of ROS before and after Lnc-CLSTN2-1:1 knockdown. The results showed that both TrxR and GPx activities were reduced after Lnc-CLSTN2-1:1 knockdown, resulting in the inhibition of antioxidant stress levels, while intracellular ROS levels were high, which eventually caused killing effects on tumor cells due to the imbalance between oxidative stress and antioxidant stress. Conclusion: Our results showed that Lnc-CLSTN2-1:1 enhanced anti-oxidative stress TrxR and GPx selenoprotein activities through the PI3K/AKT signaling pathway while counteracting the loss of reactive oxygen species ROS produced by mitochondria to osteosarcoma cells, which protected osteosarcoma cells and thus promoted the proliferation and metastatic ability of OS.

The IL-17-IL-17RA axis is required to promote osteosarcoma progression in mice

Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.

The function and mechanism of PSMD14 in promoting progression and resistance to anlotinib in osteosarcoma

BackgroundOsteosarcoma is a rare bone malignancy that frequently affects adolescents and poses formidable obstacles in its advanced stages. Studies revealed that PSMD14 may be a viable osteosarcoma treatment target. However, PSMD14’s function and mechanism in osteosarcoma remain unknown. This study aimed to examine the function and mechanism of PSMD14 in the biological behavior of osteosarcoma and its role in anlotinib resistance.MethodsWestern blotting, qRT-PCR, and immunohistochemistry (IHC) studies were used to examine PSMD14 levels. The role of PSMD14 in the malignant phenotype of osteosarcoma and its molecular pathway was explored by a series of studies, including Western blotting, cell amplification assay, transwell assay, and tumor growth. Furthermore, a series of in vitro investigations were done to determine the effect of PSMD14 on anlotinib-resistant osteosarcoma cell lines.ResultsPSMD14 expression was elevated in osteosarcoma tissues compared to normal tissues. Overexpression of PSMD14 was associated with osteosarcoma patients’ pathological grade and clinical stage, and PSMD14 was an independent poor prognostic factor. PSMD14 knockdown inhibits in vitro cell proliferation, migration, invasion, and in vivo tumor growth. PSMD14 knockdown has the potential to downregulate the PI3K/Akt/mTOR pathway, which was regarded as one of the key mechanisms promoting tumor growth. PSMD14 was likewise overexpressed in anlotinib-resistant OS cell lines, and its knockdown not only reduced the proliferation, migration, and invasion of subline cells and triggered cell apoptosis. Importantly, combination therapy with anlotinib enhanced these effects.ConclusionsPSMD14 is substantially expressed in osteosarcoma and may be an independent risk factor associated with poor prognosis. It can promote tumor progression and anlotinib resistance in osteosarcoma and may promote osteosarcoma progression by modulating PI3K/AKT/mTOR signaling pathway.

Circular RNA CircSATB2 facilitates osteosarcoma progression through regulating the miR-661/FUS-mediated mRNA of ZNFX1

Circular RNAs (circRNAs) are a class of non-coding RNAs which take part in the regulation of the initiation and development of different types of cancer. Numerous studies have demonstrated that circRNAs are involved in the progression of osteosarcoma (OS) as well. Thus, we put our emphasis on the exploration of crucial circRNAs in the process of OS initiation and progression. Using RNA sequencing, we found that circSATB2 was highly expressed in OS tissues compared with adjacent normal tissues. Then, we confirmed the high expression of circSATB2 in OS cell lines and OS tissues and its high expression was related to poor prognosis of OS patients. Functional experiments exhibited that circSATB2 promoted OS proliferation and migration in vitro, primary OS model and OS lung metastasis model showed that circSATB2 aggravated OS progression in vivo. Mechanistically, circSATB2 was found to promote OS progression through sponging miR-661 and FUS regulating the mRNA of ZNFX1. Therefore, circSATB2 could act as a prognostic marker and a therapeutic target for osteosarcoma in the future.

METTL3-mediated m6A modification of circRNF220 modulates miR-330-5p/survivin axis to promote osteosarcoma progression

BackgroundCircular RNAs (circRNAs) play a crucial role in regulating various physiological processes. However, the precise regulatory mechanisms of circRNF220s in osteosarcoma (OS) are not well understood.MethodsThe abundances of circRNF220, miR-330-5p, and survivin were determined using qRT-PCR. To assess the m6A accumulation in circRNF220, a methylated RNA immunoprecipitation (Me-RIP) assay was conducted. Cellular multiplication, motility, and invasion were examined using the cell Counting Kit-8 (CCK-8), EdU, colony formation, Transwell, and wound-healing assays. The binding relationships were measured through RNA immunoprecipitation (RIP) and luciferase reporter assays. In vivo functionality was assessed using xenograft models.ResultsCircRNF220 was identified as being overexpressed in both OS cells and tissues. In vitro experiments demonstrated that silencing circRNF220 impeded the proliferation, invasion, and motility of OS cells. Similarly, in vivo studies confirmed that downregulating circRNF220 inhibited the growth of OS. Further mechanistic investigations unveiled that METTL3-modulated circRNF220 regulated the progression of OS by upregulating survivin expression through acting as a sponge for miR-330-5p.ConclusionThe modulation of METTL3-regulated circRNF220 has been found to promote the progression of OS by modulating the miR-330-5p/survivin axis. This novel finding suggests a potentially unique approach to managing OS.

Circular RNA circBLNK promotes osteosarcoma progression and inhibits ferroptosis in osteosarcoma cells by sponging miR‑188‑3p and regulating GPX4 expression.

As a newly identified circular RNA (circRNA), the role of circBLNK in cancer progression has not been probed. The objective of the present study was to functionally dissect the role of circBLNK in osteosarcoma (OS) tumorigenesis and progression. With regards of the experimental procedure, the levels of mRNAs and proteins were assessed using reverse transcription‑quantitative PCR and western blot analysis, respectively. The subcellular location of circBLNK in OS cells was determined by cell cytosolic/nuclear fractionation assay. Cell ferroptosis ability was assessed through MTT assay. Cell proliferative abilities were assessed by clonogenic and Cell Counting Kit‑8 assays, and cell apoptosis was measured using flow cytometry. The relationships among circBLNK, miR‑188‑3p, and glutathione peroxidase 4 (GPX4) were validated by luciferase reporter and RNA pull‑down assays, as well as RNA immunoprecipitation. The stability of circBLNK and linear BLNK was confirmed using RNase R treatment assay. The association between circBLNK expression and overall survival rate was assessed by Kaplan‑Meier plot. The correlation between the expression levels of circBLNK, miR‑188‑3p, and GPX4 in OS tissues was assessed by Pearson's χ2 test. The results revealed that CircBLNK and GPX4 were significantly upregulated in OS tissues, which predicted the poor prognosis. CircBLNK knockdown led to suppressed cell proliferation and enhanced cell apoptosis, an effect that could be reversed by the inhibition of miR‑188‑3p. In an invivo circBLNK deficiency model, tumor growth was observed to be markedly suppressed. Moreover, circBLNK deficiency elevated levels of intracellular free iron (Fe2+), malondialdehyde, lipid reactive oxygen species and mitochondrial superoxide, while diminishing mitochondrial membrane potential in Erastin‑treated OS cells, which were eliminated by overexpressing GPX4. Furthermore, mechanistic investigations revealed that circBLNK sponged miR‑188‑3p to regulate the expression of GPX4, thereby affecting OS progression. In conclusion, the present study delineated a new regulatory axis involving circBLNK/miR‑188‑3p/GPX4 in OS progression, adding to the growing evidence that circRNAs are critical gene regulators in cancer progression.

Long noncoding RNA TUG1 promotes malignant progression of osteosarcoma by enhancing ZBTB7C expression

BackgroundDysregulation of long non-coding RNAs (lncRNAs) is an important component of tumorigenesis. Aberrant expression of lncRNA taurine upregulated gene 1 (lncTUG1) has been reported in various tumors; however, its precise role and key targets critically involved in osteosarcoma (OS) progression remain unclear. MethodsThe expression profiles of lncRNAs and their regulated miRNAs related to OS progression were assessed by bioinformatics analysis and confirmed by qRT-PCR of OS cells. The miRNA targets were identified by transcriptome sequencing and verified by luciferase reporter and RNA pull-down assays. Several in vivo and in vitro approaches, including CCK8 assay, western blot, qRT-PCR, lentiviral transduction and OS cell xenograft mouse model were established to validate the effects of lncTUG1 regulation of miRNA and the downstream target genes on OS cell growth, apoptosis and progression. ResultsWe found that lncTUG1 and miR-26a-5p were inversely up or down-regulated in OS cells, and siRNA-mediated lncTUG1 knockdown reversed the miR-26a-5p down-regulation and suppressed proliferation and enhanced apoptosis of OS cells. Further, we identified that an oncoprotein ZBTB7C was also upregulated in OS cells that were subjected to lncTUG1/miR-26a-5p regulation. More importantly, ZBTB7C knockdown reduced the ZBTB7C upregulation and ZBTB7C overexpression diminished the anti-OS effects of lncTUG1 knockdown in the OS xenograft model. ConclusionsOur data suggest that lncTUG1 acts as a miR-26a-5p sponge and promotes OS progression via up-regulating ZBTB7C, and targeting lncTUG1 might be an effective strategy to treat OS.