Molecular Mechanism Of Osteosarcoma Research Articles

LINC00629 protects osteosarcoma cell from ER stress-induced apoptosis and facilitates tumour progression by elevating KLF4 stability

BackgroundEscaping from ER stress-induced apoptosis plays an important role in the progression of many tumours. However, its molecular mechanism in osteosarcoma remains incompletely understood.MethodsThe molecular mechanism was investigated using RNA sequencing, qRT–PCR and Western blot assays. The relationship between LINC00629 and KLF4 was investigated using RNA pulldown and ubiquitylation assays. The transcriptional regulation of laminin subunit alpha 4 (LAMA4) by KLF4 was identified using bioinformatic analysis, a luciferase assay, and a chromatin immunoprecipitation assay.ResultsHere, we demonstrated that LINC00629 was increased under ER stress treatment. Elevated LINC00629 inhibited ER stress-induced osteosarcoma cell apoptosis and promoted clonogenicity and migration in vitro and in vivo. Further mechanistic studies indicated that LINC00629 interacted with KLF4 and suppressed its degradation, which led to a KLF4 increase in osteosarcoma. In addition, we also found that KLF4 upregulated LAMA4 expression by directly binding to its promoter and that LINC00629 inhibited ER stress-induced apoptosis and facilitated osteosarcoma cell clonogenicity and metastasis by activating the KLF4-LAMA4 pathway.ConclusionCollectively, our data indicate that LINC00629 is a critical long noncoding RNA (lncRNA) induced by ER stress and plays an oncogenic role in osteosarcoma cell by activating the KLF4-LAMA4 axis.

Screening and Analysis of Biomarkers in the miRNA-mRNA Regulatory Network of Osteosarcoma.

Osteosarcoma is a malignant disease, and few effective strategies can completely overcome the prognosis of these patients. This study attempted to reveal the key factors and related molecular mechanisms of osteosarcoma via excavating public microarray datasets. The data were obtained from the Gene Expression Omnibus (GEO) database; the differentially expressed miRNAs and differentially expressed genes were obtained in GSE69470 and GSE12685l, respectively; the target of miRNAs were predicted with the miRDIP database; the functions of the factors were analyzed and visualized by the David database and R language, respectively. Moreover, the protein-protein interaction network and miRNA-mRNA network were performed with the STRING database and Cytoscape software to identify the hub nodes in GSE69470 and GSE12685. The results showed that 834 DEGs were found in GSE12685 and 37 miRNAs were found in GSE69470. Moreover, the target of 37 miRNAs were enriched in PI3K/AKT, P53, Wnt/β-catenin, and TGF-β pathways and related with skeletal system development and cell growth. Besides, the miRNAs including miR-22-3p, miR-154-5p, miR-34a-5p, miR-485-3p, miR-93-5p, and miR-9-5p and the genes including LEF1, RUNX2, CSF1R, CDKN1A, and FBN1 were identified as the hub nodes via network analysis. In conclusion, this study suggested that the miRNAs including miR-22-3p, miR-154-5p, miR-34a-5p, miR-485-3p, miR-93-5p, and miR-9-5p and the genes including LEF1, RUNX2, CSF1R, CDKN1A, and FBN1 act as key factors in the progression of osteosarcoma.

Circ_0001174 facilitates osteosarcoma cell proliferation, migration, and invasion by targeting the miR-186-5p/MACC1 axis

BackgroundStudies of aberrantly expressed circular RNAs (circRNAs) can provide insights into the molecular mechanisms of osteosarcoma (OS). However, the role of circ_0001174 in OS progression remains unknown. This study is aimed to identify differentially expressed circRNAs and messenger RNAs (mRNAs) in patients with OS and to investigate potential regulatory ways of circ_0001174.MethodsHigh-throughput sequencing was performed to screen aberrantly expressed circRNAs and mRNAs between tumor and paracancerous tissues from patients with OS. Several bioinformatics tools were used to analyze the functions and pathways of the differentially expressed genes between the tissues. Cell counting kit-8, cell migration and invasion assays were performed to evaluate the functions of the critical circRNAs. RNA interference experiments, quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting were used to explore the relationship between miR-186-5p and circ_0001174 or metastasis-associated in colon cancer 1 (MACC1).ResultsCompared with the paracancerous tissues, 109 circRNAs and 1264 mRNAs were differentially expressed in the OS tissues, including 88 circRNAs and 707 mRNAs that were upregulated and 21 circRNAs and 557 mRNAs that were downregulated. The expression of four upregulated and four downregulated circRNAs was validated using RT-qPCR; the results were consistent with the sequencing data, and circ_0001174 was found to be significantly upregulated in 16 pairs of OS tissues and OS cell lines (fold change > 2.0, P value < 0.05). Knockdown of circ_0001174 inhibited the proliferation, migration, and invasion of OS cells. Additionally, circ_0001174 directly and negatively modulated the expression of miR-186-5p and positively regulated the expression of MACC1.ConclusionsAbnormally high expression of circ_0001174 may promote the proliferation, migration, and invasion of OS cells through up-regulating MACC1 by sponging miR-186-5p. These results provide insight into therapeutic targets for preventing and treating OS.

Circular RNA circ_UBAP2 facilitates the progression of osteosarcoma by regulating microRNA miR-637/high-mobility group box (HMGB) 2 axis

ABSTRACT Circular RNA circ_UBAP2 has been reported to be closely associated with various tumors. The present work focused on exploring the roles of circ_UBAP2 and its molecular mechanism in osteosarcoma (OS). Circ_UBAP2, miR-637, and high-mobility group box (HMGB) 2 levels in OS cells and tissues were detected by quantitative real-time polymerase chain reaction. The relationship between miR-637 and circ_UBAP2, as well as between miR-637 and HMGB2, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), Transwell and flow cytometry assays, respectively. HMGB2 protein levels were measured using Western blotting. Xenograft tumor formation assay was also performed. Circ_UBAP2 showed high expression levels in OS tissues and cells, which was directly proportional to metastasis and clinical stage of OS. The overexpression of circ_UBAP2 enhanced the growth, invasion, and migration of OS cells, but suppressed their apoptosis. In contrast, circ_UBAP2 silencing had opposite effects. Furthermore, miR-637 served as a downstream target of circ_UBAP2, which played opposite roles to circ_UBAP2 in OS. More importantly, HMGB2 served as miR-637ʹs downstream target. The xenograft experiments in nude mice also proved that knockdown of circ_UBAP2 could increase miR-637 expression, but decrease HMGB2 expression, thus alleviating OS progression. Mechanistically, circ_UBAP2 exerts a cancer-promoting effect on OS by downregulating miR-637 and upregulating the expression of HMGB2. Circ_UBAP2 plays a promoting role in OS, and the circ_UBAP2/miR-637/HMGB2 axis is involved in OS progression.

M6A-dependent upregulation of TRAF6 by METTL3 is associated with metastatic osteosarcoma

ObjectivesRNA N6-methyladenosine (m6A) is associated with tumorigenesis. The importance of methyltransferase-like 3 (METTL3) has been reported in cancer progression and metastasis. However, its role and molecular mechanism in osteosarcoma (OS), the most common primary bone tumor, is poorly studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the metastasis of OS. MethodsThe expression differences of METTL3 between metastatic and non-metastatic OS tissues and patients with different Enneking stages were detected using RT-qPCR. METTL3 was artificially downregulated in the cells, followed by wound healing assay, Matrigel assay, immunofluorescence, in vivo tumorigenic assay, HE staining, and western blot. Transcriptome sequencing and m6A-seq was conducted to identify the downstream genes of METTL3, and RIP and dual-luciferase assays were performed for validation. The expression of TRAF6 in OS tissues was detected using RT-qPCR. Finally, the rescue experiments were conducted. ResultsMETTL3 was overexpressed in metastatic OS tissues, and downregulation of METTL3 decreased cell migration, invasion, epithelial-mesenchymal transition, and tumorigenic and metastatic activities. The m6A site was highly enriched in cells poorly expressing METTL3, and the m6A peak was mainly enriched in the exon region. METTL3 was positively correlated with TRAF6 in metastatic OS, and depletion of METTL3 resulted in the loss of TRAF6 expression in OS cells. Upregulation of TRAF6 contributed to metastases in vitro and in vivo. ConclusionMETTL3 is highly expressed in OS and enhances TRAF6 expression through m6A modification, thereby promoting the metastases of OS cells.

Identification of Key Genes and Pathways in Osteosarcoma by Bioinformatics Analysis.

Purpose Osteosarcoma (OS) is the most primary bone malignant tumor in adolescents. Although the treatment of OS has made great progress, patients' prognosis remains poor due to tumor invasion and metastasis. Materials and Methods We downloaded the expression profile GSE12865 from the Gene Expression Omnibus database. We screened differential expressed genes (DEGs) by making use of the R limma software package. Based on Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis, we performed the function and pathway enrichment analyses. Then, we constructed a Protein-Protein Interaction network and screened hub genes through the Search Tool for the Retrieval of Interacting Genes. Result By analyzing the gene expression profile GSE12865, we obtained 703 OS-related DEGs, which contained 166 genes upregulated and 537 genes downregulated. The DEGs were primarily abundant in ribosome, cell adhesion molecules, ubiquitin-ubiquitin ligase activity, and p53 signaling pathway. The hub genes of OS were KDR, CDH5, CD34, CDC42, RBX1, POLR2C, PPP2CA, and RPS2 through PPI network analysis. Finally, GSEA analysis showed that cell adhesion molecules, chemokine signal pathway, transendothelial migration, and focal adhesion were associated with OS. Conclusion In this study, through analyzing microarray technology and bioinformatics analysis, the hub genes and pathways about OS are identified, and the new molecular mechanism of OS is clarified.

Synergistic effect of growth factor receptor-bound protein 2/epidermal growth factor receptor dual-targeting peptide inhibitor and salinomycin on osteosarcoma.

The growth factor receptor-bound protein 2 (Grb2)-Sos1 interaction, mediated by modular domains, plays an essential role in the oncogenic MAPK signaling pathway in osteosarcoma (OS). Recently, a dual-targeting peptide that targets the epidermal growth factor receptor and Grb2-Src homology 3 domain in OS cells was designed and synthesized. We investigated the synergistic effects of the peptide and salinomycin (Sal), a chemotherapeutic drug with effective anti-OS properties in clinical therapy. Flow cytometry was used to measure the targeting efficacy of the peptide. Migration and CCK-8 assays were used to explore whether Sal and the peptide could synergistically inhibit OS cell behavior. Western blotting was used to detect apoptosis. Data were analyzed using the GraphPad Prism 5.01. Statistical analysis was performed using the Student's t-test for the direct comparisons and one-way analysis of variance for the comparisons among the multiple groups. Statistical significance was set at P < 0.05. The peptide was shown to target OS cells. When applied together, Sal and the peptide synergistically inhibited OS cell migration, invasion, and proliferation through the inhibition of Grb2-Sos1. This synergistic treatment also promoted the apoptosis of OS cells and inhibited tumor volume in vivo. These data provide valuable insights into the molecular mechanisms of OS and may be beneficial in clinical therapy.

CCAR2 promotes a malignant phenotype of osteosarcoma through Wnt/β-catenin-dependent transcriptional activation of SPARC

CCAR2 plays a pivotal role in the regulation of the DNA damage response and cancer progression. Although aberrant expression of CCAR2 has been reported in several types of cancer, its biological function and molecular mechanism in osteosarcoma (OS) have not yet been fully elucidated. Here, we show that silence of CCAR2 prevented the malignant phenotype of OS cell in vitro and decreased tumor growth in nude mice. By analyzing the transcriptomic profile of CCAR2 knockdown U2OS cells, we identified secreted protein acidic and rich in cysteine (SPARC) is tightly regulated by CCAR2. Mechanically, we found that SPARC is transcriptionally regulated by Wnt/β-catenin signaling, and CCAR2 acts as a co-activator of Wnt/β-catenin signaling to regulate the expression of SPARC in OS cells. Additionally, SPARC knockdown largely eliminated the malignant phenotype induced by CCAR2 overexpression and forced expression of SPARC promoted the malignant phenotype of CCAR2-depleted cells. In conclusion, our results suggest that CCAR2 exerted oncogenic roles in OS cells mainly via up-regulating SPARC expression and targeting the CCAR2-SPARC axis might have promising application prospect for the treatment of osteosarcoma.

MicroRNA-1226-3p has a tumor-promoting role in osteosarcoma.

Osteosarcoma is a malignant bone tumor that commonly occurs in young individuals. It accounts for 10% of solid tumors in those who are 15–19 years old. MicroRNA (miRNA/miR) dysregulation serves a crucial role in the molecular mechanism of osteosarcoma. The present study reported a novel miRNA (miR-1226-3p) and investigated its function in osteosarcoma. miR-1226-3p mimics and miR-1226-3p antisense oligonucleotides were transfected into human osteosarcoma SaOS-2 cells to alter miR-1226-3 expression, while the hFOB 1.19 cell line was used as the control. The apoptosis rate was analyzed using a dead cell apoptosis kit. TNF receptor-associated factor 3 (TRAF3) protein expression was assayed by western blotting. The results of bioinformatics and clinical specimen analyses revealed that higher expression levels of miR-1226-3p were associated with lower survival rates. Additionally, the results of experiments on cultured cells revealed that miR-1226-3p promoted the proliferation of SaOS-2 cells, while miR-1226-3p inhibition decreased cell proliferation and increased apoptosis. Furthermore, it was revealed that miR-1226-3p targeted TRAF3 in SaOS-2 cells. In conclusion, the present study suggested that miR-1226-3p promoted the proliferation of osteosarcoma cells.

The miR-193a-5p/NCX2/AKT axis promotes invasion and metastasis of osteosarcoma.

MiR-193a-5p has been observed to have oncogenic or tumor suppressive functions in different kinds of cancers, but its role and molecular mechanism in osteosarcoma are elusive. Na+/Ca2+ exchangers (NCX1, NCX2 and NCX3) normally extrude Ca2+ from the cell, and deregulation of the intracellular Ca2+ homeostasis is related to several kinds of diseases, including cancer. The present study demonstrated that miR-193a-5p was upregulated in osteosarcoma tissues compared with the corresponding adjacent noncancerous tissues, and promoted colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) in osteosarcoma cells (SaOS-2 and U-2OS), as well as metastasis in a murine xenograft model. Tandem mass tag-based quantitative proteomics analysis identified NCX2 as a potential target of miR-193a-5p. Luciferase activity assays and Western blotting further confirmed that miR-193a-5p recognized the 3′-untranslated region of NCX2 mRNA, and negatively regulated NCX2 expression. NCX2 was downregulated in osteosarcoma tissues, and its expression was negatively correlated with miR-193a-5p levels. Ectopic expression of NCX2 in osteosarcoma cells could reverse the oncogenicity of miR-193a-5p, indicating that miR-193a-5p exerted its effects by targeting NCX2. Further study demonstrated that NCX2 suppresses Ca2+-dependent Akt phosphorylation by decreasing intracellular Ca2+ concentration, and then inhibited EMT process. Treatment with the antagomir against miR-193a-5p sensitized osteosarcoma to the Akt inhibitor afuresertib in a murine xenograft model. In conclusion, a miR-193a-5p/NCX2/AKT signaling axis contributes to the progression of osteosarcoma, which may provide a new therapeutic target for osteosarcoma treatment.

Notch-1 promotes the malignant progression of osteosarcoma through the activation of cell division cycle 20.

The molecular mechanism of osteosarcoma (OS) pathogenesis is poorly understood. The Notch signaling pathway has been shown to be critically involved in tumorigenesis, including OS. Therefore, we explored the molecular mechanism by which the Notch-1 signaling pathway is involved in OS progression. Several approaches were carried out to determine the biological function of Notch-1 in OS cells. The MTT results revealed that Notch-1 overexpression increased the viability of OS cells, whereas Notch-1 downregulation reduced cell viability. Consistently, modulation of Notch-1 regulated apoptosis and the migratory and invasive abilities of OS cells. Mechanistic studies showed that Notch-1 overexpression augmented cell division cycle 20 (Cdc20) expression in OS cells. Moreover, overexpression of Cdc20 alleviated the inhibitory effects of Notch-1 downregulation on the viability, migration and invasion of OS cells. Our study offers a promising OS treatment strategy by inhibiting Notch-1.

Molecular Characterisation of Canine Osteosarcoma in High Risk Breeds.

Dogs develop osteosarcoma (OSA) and the disease process closely resembles that of human OSA. OSA has a poor prognosis in both species and disease-free intervals and cure rates have not improved in recent years. Gene expression in canine OSAs was compared with non-tumor tissue utilising RNA sequencing, validated by qRT-PCR and immunohistochemistry (n = 16). Polymorphic polyglutamine (polyQ) tracts in the androgen receptor (AR/NR3C4) and nuclear receptor coactivator 3 (NCOA3) genes were investigated in control and OSA patients using polymerase chain reaction (PCR), Sanger sequencing and fragment analysis (n = 1019 Rottweilers, 379 Irish Wolfhounds). Our analysis identified 1281 significantly differentially expressed genes (>2 fold change, p < 0.05), specifically 839 lower and 442 elevated gene expression in osteosarcoma (n = 3) samples relative to non-malignant (n = 4) bone. Enriched pathways and gene ontologies were identified, which provide insight into the molecular pathways implicated in canine OSA. Expression of a subset of these genes (SLC2A1, DKK3, MMP3, POSTN, RBP4, ASPN) was validated by qRTPCR and immunohistochemistry (MMP3, DKK3, SLC2A1) respectively. While little variation was found in the NCOA3 polyQ tract, greater variation was present in both polyQ tracts in the AR, but no significant associations in length were made with OSA. The data provides novel insights into the molecular mechanisms of OSA in high risk breeds. This knowledge may inform development of new prevention strategies and treatments for OSA in dogs and supports utilising spontaneous OSA in dogs to improve understanding of the disease in people.

Comprehensive Characterization of Prognostic Long Noncoding RNAs in Osteosarcoma.

The molecular mechanism of osteosarcoma (OS) based on protein-coding genes has largely been studied in the past decades. However, much remains to be explored when it comes to the role that long noncoding RNAs (lncRNAs) play in the pathogenesis and progression of OS and how they are associated with OS metastasis. In the present study, we collected RNA-seq-based gene expression data of 82 OS samples from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) database, along with their clinical information. We found that 50 lncRNAs were significantly associated with patients' survival by univariable Cox regression model. Moreover, we built multivariable Cox regression model based on 7 lncRNAs and successfully stratified patients into two risk groups, which exhibited significantly different prognostic outcomes. Significantly enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways detected by differential expression analysis on DEGs between the two groups with different prognostic outcomes were both immune-related, indicating that such GO terms and pathways are critical for OS survival. Among the seven lncRNA signatures, AC011442.1 was predicted to act as an oncogenic driver in OS by correlation analysis of copy number alteration (CNA) and lncRNA expression, and it was predicted to regulate AMPK and hedgehog signaling pathways. In summary, the identification of novel prognostic lncRNAs in OS could not only improved our understanding of the lncRNAs involved in OS tumorigenesis or progression but also assist the diagnosis and development of molecularly targeted therapies for OS, which in turn benefit patients' survival.

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.

Identification of the Novel Target Genes for Osteosarcoma Therapy Based on Comprehensive Bioinformatic Analysis.

Osteosarcoma is one of the most common primary malignant tumors of the bone and tends to develop in teenage years. Although multitreatments for the diagnosis and therapy of osteosarcoma have been developed, there are still needs of new methods to prevent and treat the osteosarcoma. Here, we performed bioinformatic analysis to screen for the key genes, molecules, and pathways involved in osteosarcoma survival. Four microarray data sets (GSE99671, GSE87624, GSE65071, and GSE28423), which include data from human bone and osteosarcoma samples, were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed mRNAs and miRNAs were identified. Kyoto Encyclopedia of Genes and Genomes enriched pathways, miRNA-mRNA target, gene/disease relationship, and overall survival was elucidated using related websites and software according to bioinformatic analysis protocols. We found three critical genes miR-29c, blood vessel epicardial substance (BVES), and proteasome 20S subunit beta 2 (PSMB2) through the GEO database and predicting miRNA-mRNA target. Among these genes, BVES and PSMB2 presented a high expression level in osteosarcoma based on GSE99671 and GSE87624 data sets, while miR-29c showed a low expression level in osteosarcoma based on GSE65071 and GSE28423 data sets. Furthermore, we found that the high expression level of miR-29c and BVES associated with better prognosis, while highly expressed PSMB2 associated with poor prognosis. The abnormally expressed mRNAs and miRNAs, which were identified by integrated bioinformatic analysis, provided insights into the molecular mechanisms of osteosarcoma. Notably, we found three critical genes that could be used as novel therapeutic targets for preventing or diagnosing osteosarcoma. Finally, PSMB2 may be the target of miR-29c.

LncRNA DANCR regulates osteosarcoma migration and invasion by targeting miR-149/MSI2 axis.

As a new LncRNA, anti-differentiated non-coding RNA (DANCR) plays an important role in tumorigenesis and development, and its molecular mechanism in osteosarcoma is unclear. In this study, by investigating osteosarcoma tissue and cells, we explored the molecular mechanism by which lncRNA DANCR regulates the occurrence and development of osteosarcoma by targeting the miR-149 / MSI2 axis. In this study, osteosarcoma tissues and adjacent tissues in 109 patients were collected, and the relative expression of DANCR was detected by qPCR. The correlation between DANCR expression and clinical classification was statistically analyzed. In order to explore the potential molecular mechanism of DANCR related to tumor migration and invasion, an overexpression and silencing test was performed on the osteosarcoma cell line Saos-2, and then qPCR method was used to test the expression of miR149, and cell scratch test was used to detect invasion after DANCR silencing and miR149 overexpression. Transwell assay was used to detect the invasion after DANCR silencing and miR149 overexpression. Finally, Western blot was used to verify the expression of MSI2 protein after overexpression and silencing of miR-149. DANCR was significantly up-regulated in both osteosarcoma tissue and cells. The high expression of DANCR was significantly positively correlated with tissue typing and advanced TNM stage. DANCR can significantly reduce the migration and invasion of osteosarcoma cells. miRNA overexpression significantly reduced osteosarcoma cell migration and invasion. When miR-149 was overexpressed, MSI2 protein expression was significantly down-regulated. When miR-149 was silenced, MSI2 protein was significantly up-regulated. LncRNA DANCR plays an important regulatory role in the occurrence and development of osteosarcoma. It may be used as a potential target in the treatment of osteosarcoma in the future, by targeting the miR-149/MSI2 axis to regulate the occurrence and development of osteosarcoma.

Silencing METTL3 inhibits the proliferation and invasion of osteosarcoma by regulating ATAD2

BackgroundOsteosarcoma is the most common primary malignant bone tumor in children and young adults. RNA N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA, which is involved in tumorigenesis and tumor progression. It has been reported that methyltransferase-like 3 (METTL3), the first reported m6A “writer”, plays critical roles in cancer progression. However, its role and molecular mechanism in osteosarcoma is poor studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the progression of osteosarcoma. MethodsWe detected the mRNA expression of METTL3 in osteosarcoma cell lines, and immunofluorescence assay was performed to observe the location of METTL3. Cell lines with METTL3 gene overexpression or knockdown were established by pcDNA3.1-METTL3 or siRNA interferences in order to determine the function of METTL3 in osteosarcoma in vitro. Transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3 in osteosarcoma. ResultsWe found that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. Silencing METTL3 in SAOS-2 and MG63 cells significantly inhibited the m6A methylation level, proliferation, migration, and invasion abilities, as well as promoted cell apoptosis. However, up-regulation of METTL3 had no significant effect on the biological behaviors of U2OS cells. Further mechanism analysis suggested that METTL3 knockdown inhibited the expression of ATPase family AAA domain containing 2 (ATAD2). Moreover, ATAD2 knockdown inhibited the proliferation and invasion of SAOS-2 and MG63 cells, while its overexpression showed a significant increase in cell proliferation and invasion. Furthermore, METTL3 knockdown abrogated the promoting effects of ATAD2 overexpression on osteosarcoma cells proliferation and invasion. ConclusionOverall, our study revealed that METTL3 functions as an oncogene in the growth and invasion of osteosarcoma by regulating ATAD2, suggesting a potential therapeutic target for osteosarcoma treatment.

LncRNA TMPO-AS1 serves as a ceRNA to promote osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis.

Osteosarcoma (OS) is a common kind of aggressive tumor in bone which was mostly identified in children and adolescents with extremely high risk of death. Accumulating research works have displayed that long noncoding RNAs (lncRNAs) exert an essential role in the development of multiple cancers. It has been reported that TMPO-AS1 is an oncogene in cancers; nonetheless, its molecular mechanism in OS is totally unclear. Our present study elucidated that a remarkable overexpression of TMPO-AS1 was found in OS tissues and cells. Moreover, TMPO-AS1 depletion restrained Wnt/β-catenin pathway and cell proliferation as well as facilitated cell apoptosis. Further molecular mechanism investigations showed that TMPO-AS1 can sponge to miR-199a-5p. Moreover, miR-199a-5p was at a low level at OS cells. Importantly, miR-199a-5p's overexpression was associated with the OS cells' decreased proliferation and increased apoptosis. In addition, WNT7B was confirmed as a downstream gene of miR-199a-5p. Also the WNT7B expression was reversely modulated by miR-199a-5p and positively modulated by TMPO-AS1. Rescue experiments suggested that downregulated WNT7B rescued miR-199a-5p inhibitor-mediated repression on OS progression, but the treatment of LiCl counteracted the effect of WNT7B downregulation. In a word, TMPO-AS1 serves as a competing endogenous RNA to boost osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis, which provided an underlying therapeutic target for patients with OS.

The proliferation and invasion of osteosarcoma are inhibited by miR-101 via targetting ZEB2.

Having a better grasp of the molecular mechanisms underlying carcinogenesis and progression in osteosarcoma would be helpful to find novel therapeutic targets. Different types of cancers have presented abnormal expression of miRNA-101 (miR-101). Nevertheless, we still could not figure out what expression of miR-101 in human osteosarcoma is and its biological function. Thus, we conducted the present study to identify its expression, function, and molecular mechanism in osteosarcoma. We detected the expression of miR-101 in osteosarcoma samples and cell lines. The effects of miR-101 on osteosarcoma cells’ proliferation and invasion were evaluated. Luciferase reporter assay was applied to identify the direct target of miR-101. Compared with adjacent normal specimens and normal bone cell line by using qPCR, the expression levels of miR-101 in osteosarcoma specimens and human osteosarcoma cell lines distinctly decreased. According to function assays, we found that overexpression of miR-101 significantly inhibited the cell proliferation and invasion in osteosarcoma cells. Moreover, we confirmed that zinc finger E-box binding homeobox 2 (ZEB2) was a direct target of miR-101. In addition, overexpression of ZEB2 could rescue the inhibition effect of proliferation and invasion induced by miR-101 in osteosarcoma cells. MiR-101 has been proved to be down-regulated in osteosarcoma and has the ability to suppress osteosarcoma cell proliferation and invasion by directly targetting ZEB2.

Long Noncoding RNA FEZF1-AS1 Promotes Osteosarcoma Progression by Regulating the miR-4443/NUPR1 Axis.

Long noncoding RNA (lncRNA) FEZF1-AS1 was demonstrated to facilitate cell proliferation and migration in some cancers. However, the functions of FEZF1-AS1 and its molecular mechanism in osteosarcoma remain to be elucidated. In our study, we found that the expression of FEZF1-AS1 was upregulated in osteosarcoma samples and cell lines compared with normal tissues or cells. Besides, we showed that the expression levels of FEZF1-AS1 in osteosarcoma patients were positively correlated with tumor metastasis and TNM stage. Additionally, FEZF1-AS1 knockdown inhibited cell proliferation, migration, and invasion in U2OS and MG63 cells, while upregulation had the opposite effects in vitro. Moreover, FEZF1-AS1 depletion inhibited tumor growth and metastasis in vivo. We found that FEZF1-AS1 sponged miR-4443 to promote NUPR1 expression in U2OS and MG63 cells. Furthermore, knockdown of miR-4443 abrogated FEZF1-AS1 silencing-induced inhibition of cell proliferation, migration, and invasion in osteosarcoma. Finally, we found that restoration of NUPR1 rescued the proliferation, migration, and invasion abilities of FEZF1-AS1-depleted U2OS and MG63 cells. Our study indicated that FEZF1-AS1 could promote osteosarcoma progression by sponging miR-4443 to promote NUPR1 expression. The FEZF1-AS1/miR-4443/NUPR1 axis may act as a novel therapeutic strategy for osteosarcoma treatment.