Human Osteosarcoma Cell Line Research Articles

Effects of Cabozantinib on Human G292 Osteosarcoma Cells

Cabozantinib (CBZ) N-(4-((6,7-dimethoxyquinolin-4-yl) oxy) phenyl)-N-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide) (XL184), an inhibitor of MET and vascular endothelial growth factor receptor (VEGFR-2), is an agent approved for the treatment of several types of carcinoma such as medullary thyroid and renal. Recent studies are encouraging for the effectiveness of CBZ in the treatment of osteosarcoma. Because of the complex nature of the microenvironment of osteosarcoma cell sites, in order to better understand the direct effects of CBZ on osteosarcoma cells, in vitro studies were conducted with the human osteosarcoma cell line, G292. Experiments were focused on the effects of CBZ on cell metabolic activity, differentiation, and apoptosis as well as the modulation of responses to growth factors such as platelet-derived growth factor (PDGF) and insulin like growth factor (IGF-I). The results indicate that CBZ can increase the activity of caspase 3/7 as an indicator of apoptosis as well as decrease cellular activity, measured by MTT assay and differentiation assessed by alkaline phosphatase activity. The drug partially downregulated the effects of PDGF on MTT activity and had significant inhibitory effects on the G292 cells response to IGF-I and production of VEGF. This study presents original information on responses of G292 human osteosarcoma cells to the chemotherapy agent, CBZ, and provides in vitro data consistent with the potential therapeutic effects of this agent for osteosarcoma.

Identification of Conserved Pappalysin 1-Derived Circular RNA-Mediated Competing Endogenous RNA in Osteosarcoma.

The etiology of osteosarcoma (OS) is complex and not fully understood till now. This study aimed to identify the miRNAs, circRNAs, and genes (mRNAs) that are differentially expressed in OS cell lines to investigate the mechanism of circRNA-associated competing endogenous RNAs (ceRNAs) in OS. Microarray datasets reporting mRNA (GSE70414), miRNA (GSE70367), and circRNA changes (GSE96964) in human OS cell lines were downloaded, differentially expressed (DE) RNAs were identified, and DEmRNAs were used for the annotation of Gene Ontology (GO) biological processes (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The mechanisms of DEcircRNA-mediated ceRNAs were identified in a step-by-step process. A total of 326 DEmRNAs, 45 DEmiRNAs, and 110 DEcircRNAs were identified from 3 datasets. The DEmRNAs were associated with GO BP terms, including cholesterol biosynthetic process, angiogenesis, extracellular matrix organization and KEGG pathways, including p53 signaling pathway and biosynthesis of antibiotics. The final ceRNA network consisted of 8 DEcircRNAs, including 5 pappalysin (PAPPA) 1-derived DEcircRNAs (hsa_circ_0005456, hsa_circ_0088209, hsa_circ_0002052, hsa_circ_0088214 and has_circ_0008792, all downregulated), 3 DEmiRNAs (hsa-miR-760, hsa-miR-4665-5p and hsa-miR-4539, all upregulated), and downregulated genes (including MMP13 and HMOX1). The ceRNA regulation network of OS was built, which played important roles in the pathogenesis of OS and might be of great importance in therapy.

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-125-5p targets Kruppel-like factor 13 (KLF13) to regulate the proliferation, migration, and invasion of human osteosarcoma cells*

Studies have reported frequent downregulation of microRNA-125-5p (miR-125) in osteosarcoma tissues. However, the molecular role and mechanism of action of miR-125-5p in osteosarcoma via regulation of Kruppel-like factor 13 (KLF13) is yet to be explored. The results of the present study miR-125-5p are significantly (P < .05) downregulated in human osteosarcoma tissues and cell lines. Overexpression of miR-125-5p resulted in a significant (P < .5) decrease of the osteosarcoma cells via induction of autophagy. Additionally, overexpression of miR-125-5p also decreased the migration and invasive potential of the osteosarcoma cells. Bioinformatic analysis and dual-luciferase assays revealed that miR-125-5p targets KLF13. Post-transcriptional suppression of KLF13 by miR-125-5p results in the inhibition of osteosarcoma cells. Nonetheless, overexpression of KLF13 could abolish the tumor-suppressive effects of miR-125-5p. Taken together, these results suggest the tumor-suppressive role of miR-125-5p in and point toward its therapeutic potential in the treatment of osteosarcoma.

In vitro cytotoxicity of biodegradable Zn-Mg surgical wires in tumor and healthy cells

In this work, we examined the in vitro cytotoxicity of new biodegradable surgical wires. The wires made of zinc with the addition of a small amount of magnesium (pure zinc, ZnMg 0.0026, ZnMg 0.0068, and ZnMg 0.08) have been investigated. The wires were produced using a technology based on extrusion and subsequent drawing. The resulting wires with a diameter of 0.8–1.0 mm are designed to be used in surgical operations related to bone joints. For cytotoxicity studies, we have selected human dental pulp stem cells (hDPSC) as the cell population representing normal osteoprogenitor cells. Considering that, after bone surgeries, the chance of osteosarcoma increases, we have compared the results obtained in hDPSC to those obtained with Saos-2 human osteosarcoma cell line. Cultured cells were exposed to the extracts obtained from the materials incubated in culture medium for 24 h with and without preincubation. Extracts of different ratios were examined. The results showed that the extracts obtained from wires made of ZnMg 0.0026 alloy exhibit high toxicity to Saos-2 osteosarcoma cells and low toxicity to hDPSC cells. This was in contrast to all reference materials, i.e., commercial surgical sutures made of steel and polymers, that did not display cytotoxicity toward osteosarcoma cells. Thus, the detected phenomenon for the ZnMg 0.0026 alloy can become the basis for creating biodegradable Zn-Mg surgical wires with antitumor activity.

Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.

Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.

Effects of Iron Chelation in Osteosarcoma.

Osteosarcoma is an aggressive bone tumor. It represents the principal cause of cancer-associated death in children. Considering the recent findings on the role of iron in cancer, iron chelation has been investigated for its antineoplastic properties in many tumors. Deferasirox is the most used iron chelator compound and in previous studies showed an anticancer effect in hematologic and solid malignancies. Eltrombopag is a Thrombopoietin receptor used in thrombocytopenia that also binds and mobilize iron. It demonstrated an effect on iron overload conditions and also in contrasting cancer cell proliferation. We analyzed the effects of deferasirox and eltrombopag in human osteosarcoma cells in an attempt to identify other therapeutic approaches for this tumor. We cultured and treated with deferasirox and Eltrombopag, alone and in combination, two human osteosarcoma cell lines, MG63 and 143B. After 72h exposure, we performed RTqPCR, Western Blotting, Iron Assay and cytofluorimetric assays to evaluate the effect on viability, apoptosis, cell cycle progression and ROS production. The iron-chelating properties of the two compounds are also confirmed in osteosarcoma, but we did not observe any direct effect on tumor progression. We tested deferasirox and eltrombopag, alone and in combination, in human osteosarcoma cells for the first time and demonstrated that their iron-chelating activity does not influence biochemical pathways related to cancer progression and maintenance. Although further investigations on possible effects mediated by cells of the tumor microenvironment could be of great interest, in vitro iron chelation in osteosarcoma does not impair tumor progression.

CircCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression

Osteosarcoma is the most common primary malignant bone tumor in adolescents. While chemotherapy combined with surgery can improve the prognosis of some patients, chemo-resistance is still a huge obstacle in osteosarcoma treatment. Accumulating evidence demonstrates that circular RNAs (circRNAs) are involved in cancer progression and metastasis, but their specific role in osteosarcoma remains mostly undescribed. In this study, we performed circRNA deep sequencing and identified 88 distinct circRNAs from a human osteosarcoma cell lines group (143B, HOS, SJSA, and U2OS) and the human osteoblast hFOB 1.19 (control). We found that circCAMSAP1, also named hsa_circ_0004338, is significantly upregulated in human osteosarcoma tissues and cell lines, and it is positively correlated with osteosarcoma development. Silencing of circCAMSAP1 effectively suppresses osteosarcoma cell growth, apoptosis, migration, and invasion. Furthermore, we validated that circCAMSAP1 functions in osteosarcoma tumorigenesis through a circCAMSAP1/miR-145-5p/friend leukemia virus integration 1 (FLI1) pathway. FLI1 promotes osteosarcoma tumorigenesis and miR-145-5p suppresses FLI translation. circCAMSAP1 directly sequesters miR-145-5p in the cytoplasm and inhibits its activity to suppress osteosarcoma tumorigenesis. Moreover, the regulatory role of circCAMSAP1 upregulation was examined and validated in rats. In summary, our findings provide evidence that circCAMSAP1 act as a “microRNA sponge” and suggest a new therapeutic target of human osteosarcoma.

Micro-RNA 122 and micro-RNA 96 affected human osteosarcoma biological behavior and associated with prognosis of patients with osteosarcoma

Osteosarcoma (OS) is the most common bone malignancy in both children and adolescents. In the present study, we aimed to explore the association of miRNA-122 and miRNA-96 expression with the clinical characteristics and prognosis of patients with osteosarcoma. The expression of miRNA-122 and miRNA-96 in human osteosarcoma cell lines and tissues were detected in the present study. Reverse transcriptase-PCR (RT-PCR) was used to determine the expression levels of miRNA-122 and miRNA-96 in 68 human OS samples. We found that MiRNA-122 and miRNA-96 were widely up-regulated in osteosarcoma, gastric cancer and pancreatic cancer. In HOS, Saos-2 and U2OS osteosarcoma cells, miRNA-122 and miRNA-96 were up-regulated significantly, while down-regulated in MG-63 cells. After further investigation, we found that miRNA-122 and miRNA-96 concentrations were significantly higher in the tumor tissues than those in the normal tissues (P<0.01). Moreover, the cell proliferation of LV-miRNA-122-RNAi and LV-miRNA-96-RNAi transfected SaOS2 was significantly decreased compared with the LV- miRNA-122-RNAi-CN and LV- miRNA-96-RNAi group. After adjusting for competing risk factors, we found combined high miRNA-122 and miRNA-96 expression was identified as independent predictor of overall survival.

Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

Replication stress and FOXM1 drive radiation induced genomic instability and cell transformation.

In contrast to the vast majority of research that has focused on the immediate effects of ionizing radiation, this work concentrates on the molecular mechanism driving delayed effects that emerge in the progeny of the exposed cells. We employed functional protein arrays to identify molecular changes induced in a human bronchial epithelial cell line (HBEC3-KT) and osteosarcoma cell line (U2OS) and evaluated their impact on outcomes associated with radiation induced genomic instability (RIGI) at day 5 and 7 post-exposure to a 2Gy X-ray dose, which revealed replication stress in the context of increased FOXM1b expression. Irradiated cells had reduced DNA replication rate detected by the DNA fiber assay and increased DNA resection detected by RPA foci and phosphorylation. Irradiated cells increased utilization of homologous recombination-dependent repair detected by a gene conversion assay and DNA damage at mitosis reflected by RPA positive chromosomal bridges, micronuclei formation and 53BP1 positive bodies in G1, all known outcomes of replication stress. Interference with the function of FOXM1, a transcription factor widely expressed in cancer, employing an aptamer, decreased radiation-induced micronuclei formation and cell transformation while plasmid-driven overexpression of FOXM1b was sufficient to induce replication stress, micronuclei formation and cell transformation.

PolyphyllinVII induces apoptosis and autophagy via mediating H2O2 levels and the JNK pathway in human osteosarcoma U2OS cells.

Polyphyllin VII, a compound extracted from the rhizomes of Paris polyphylla, has strong antitumor effects on various human tumor cell lines. However, few studies have reported the possible effect of Polyphyllin VII on human osteosarcoma (OS) cell lines. The present study revealed that Polyphyllin VII promoted OS cell apoptosis and inhibited cell proliferation via upregulating the expression of LC3II, Atg5, Atg7 and the Atg12-Atg5 complex. By contrast, treatment of OS cells with Polyphyllin VII downregulated Atg12 and p62 expression. Following treatment with class III PI 3-kinase inhibitor (3-MA; an autophagy inhibitor), the Polyphyllin VII-mediated apoptotic effect was reversed. These findings indicated that the inhibition of autophagy could attenuate U2OS cell apoptosis in cells treated with high concentrations of Polyphyllin VII. The present study also demonstrated that Polyphyllin VII upregulated the intracellular hydrogen peroxide (H2O2) levels in U2OS cells. However, treatment of U2OS cells with N-acetyl-L cysteine (NAC) effectively reversed this effect. The western blot analysis results indicated that the c-Jun N-terminal kinase (JNK) signaling pathway was closely associated with Polyphyllin VII-induced apoptosis and autophagy. In conclusion, the results of the present study demonstrated that Polyphyllin VII could effectively inhibit cell viability and promote autophagy and apoptosis in U2OS cells. In addition, the mechanism underlying these effects could be associated with the intracellular H2O2 levels and the JNK signaling pathway.

IGFBP-3 stimulates human osteosarcoma cell migration by upregulating VCAM-1 expression

AimsInsulin-like growth factor (IGF) signaling has been documented in several human malignancies and is thought to contribute to cellular differentiation and migration, as well as malignant progression. A major binding molecule of IGF, IGF-binding protein 3 (IGFBP-3), regulates multiple IGF effects. Here, we focused on the effect of IGFBP-3 in the motility of osteosarcoma cells and examined signaling regulation. Materials and methodsUsing a human osteosarcoma tissue array, immunohistochemical staining determined levels of IGFBP-3 expression in osteosarcoma tissue and in normal tissue. The wound healing migration assay, Transwell migration assay, luciferase reporter assay, immunofluorescence staining, Western blot and real-time quantitative PCR were performed to examine whether IGFBP-3 facilitates VCAM-1-dependent migration of osteosarcoma cells. Key findingsIn this study, we found significantly higher IGFBP-3 levels in osteosarcoma tissue compared with normal healthy tissue. IGFBP-3 treatment of two human osteosarcoma cell lines promoted cell migration and upregulated levels of VCAM-1 expression via PI3K/Akt and AP-1 signaling. SignificanceIGFBP-3 appears to be a novel therapeutic target in metastatic osteosarcoma.

Neuropilin and tolloid-like 2 regulates the progression of osteosarcoma.

Neuropilin and tolloid-like 2 (NETO2) is aberrantly expressed in various malignancies. However, its role in osteosarcoma (OS) remains to be elucidated. This study aimed to identify the function of NETO2 in OS cells. The expression of NETO2 in sarcoma tissues was determined using the GEPIA database, and the mRNA and protein expression of NETO2 in OS cells and OS tissue was also assessed. The biological effects of NETO2 on OS cells were determined by overexpressing and downregulating NETO2. Cell proliferation, invasion, migration, colony formation, and epithelial-mesenchymal transition in OS cells were evaluated. Consistent with the GEPIA database, expression of NETO2 was upregulated in human OS samples and cell lines. NETO2 overexpression not only promoted the proliferation, colony formation, invasion, and epithelial-mesenchymal transition of OS cells, but also activated the PI3K/AKT signaling. NETO2 downregulation resulted in opposite effects. Furthermore, after using an AKT inhibitor, the effects of NETO2 on OS cells were attenuated. In conclusion, this study showed that NETO2 functions as an oncogene of osteosarcomas by activating the PI3K/AKT pathway.

Anti-tumour effect of tocilizumab for osteosarcoma cell lines.

AimsTocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines.MethodsWe used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay.ResultsThe OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2.ConclusionIn conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells.Cite this article: Bone Joint Res 2020;9(11):821–826.

Downregulation of CDC20 suppressed cell proliferation, induced apoptosis, triggered cell cycle arrest in osteosarcoma cells, and enhanced chemosensitivity to cisplatin.

Osteosarcoma (OS) is a common malignant bone tumor that occurs in adolescents or children under the age of 20, which is extremely difficult to cure and has a high recurrence rate. Recent studies showed that cell division cycle 20 (CDC20) overexpression is associated with poor prognosis in patients with osteosarcoma. However, the function of CDC20 in osteosarcoma has not been investigated clearly. In this study, we aim to explore the role of CDC20 in two independent human OS cell lines' biological phenotype and chemotherapy sensitivity. We applied multiple approaches to measure cell growth, cell cycle, and apoptosis with or without deregulation or overexpression of CDC20. We found that the downregulation of CDC20 by siRNA or apcin suppressed cell proliferation, induced apoptosis, and triggered cell cycle arrest. Consistently, overexpression of CDC20 in normal cells promoted cell growth, inhibited apoptosis. What's more, the additional treatment with siCDC20 or Apcin achieved better anticancer effects than that of cisplatin alone. Furthermore, Bim and p21 were upregulated in OS cells following Apcin treatment. Altogether, the results of the present study demonstrated that targeting CDC20 could be useful for the treatment of OS, and might be a promising solution for the treatment of the OS with cisplatin insensitivity.

Developing a New Beta‐Type of Ti–Si/Sn Alloys for Targeted Orthopedic Therapeutics: Assessments of Biological Characteristics

Titanium (Ti) alloys are widely used in tissue engineering, but their applications are limited by low strength, bactericidal properties, and metal ion release. Beta Ti alloys are promising materials for load‐bearing orthopedic implants due to their excellent corrosion resistance and high biocompatibility. Herein, developed new beta‐Ti alloys including Ti–Nb–Zr–Sn (Ti–Sn) and Ti–Nb–Zr–Ta–Si (Ti–Si) to improve structural and biochemical features of the existing Ti alloys as orthopedic implants. The new Ti–Sn and Ti–Si alloys were fabricated using mechanical ball milling and spark plasma sintering techniques respectively and compared with commercially pure Ti alloys on hydrophilicity, surface characteristics and cytotoxicity, proliferative and osteogenic differentiation effects as well as biocompatibility in human bone osteosarcoma cell line (MG63). The new beta Ti alloys showed no significant differences in proliferation and cytotoxicity on the MG63 cells. Both Ti–Sn and Ti–Si alloys, compared to the commercial pure Ti, improved the biological profile and upregulated the expressions of runt‐related transcription factor 2, osterix, and collagen mRNA levels in the MG63 cells. The Ti–Si alloy showed greater cell proliferation and osteoblastic protein expression and a better biological profile compared to the Ti and Ti–Sn alloys. The novel beta Ti alloys exhibited promising potentials in orthopedic applications.

Direct Measurement of the Intracellular Concentration of 8-Oxo-2'-Deoxyguanosine-5'-Triphosphate by LC-MS/MS

Exposure to reactive oxygen species can result in formation of oxidized nucleotides which increase the frequency of mutations, DNA damage, and cell death. MutT Homolog 1 (MTH1) is an enzyme that catalyzes removal of pyrophosphate from the highly mutagenic oxidized nucleoside triphosphate 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Interest in MTH1 as a potential new target for cancer therapy surged due to reports of MTH1 inhibitors causing DNA damage and inducing cell death in cancer cells. However, questions have been raised about MTH1 target validation. One critical piece of information that is currently lacking is a quantitative understanding of the levels of 8-oxo-dGTP in cancer cells and the effect of MTH1 inhibition on them. In this study, we developed a sensitive and selective method to simultaneously measure the intracellular concentrations of 8-oxo guanosine nucleotides and their unmodified counterparts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS). An ion-pairing reversed phase liquid chromatography method using dimethylhexylamine was employed to quantify the highly polar analytes. The method was validated fit for purpose using a combination of authentic standards and cell samples. The intracellular oxo-nucleotide concentrations in human bone osteosarcoma cell line U2OS were determined using the method. We observed very low levels of 8-oxo nucleotides in untreated and hydrogen peroxide treated cells and MTH1 knockdown in these cells had minimal if any effect on 8-oxo nucleotide levels. This method will allow for a more comprehensive understanding of oxidized nucleotide detoxification pathways and MTH1 as a target for cancer therapy.

Long noncoding RNA KCNQ1OT1 contributes to tumor growth and activates Wnt/β‑catenin signaling inosteosarcoma by targeting the miR‑3666/KLF7 axis.

Osteosarcoma (OS) is a type of primary malignant cancer occurring in the bone and poses a threat to the lives of children and young adults. Long non‑coding RNAs (lncRNAs) have been certified to play important roles in various human malignant tumors, including OS. lncRNA KCNQ1OT1 has been investigated in certain types of cancer; however, its role and molecular mechanisms in OS remain to be determined. In the present study, a high KCNQ1OT1 expression was detected in human OS tissues and cell lines. Moreover, patients with OS with a high expression of KCNQ1OT1 presented a worse prognosis. Loss‑of‑function assays demonstrated that KCNQ1OT1 silencing suppressed cell proliferative, migratory and invasive abilities in OS. Importantly, the knockdown of KCNQ1OT1 suppressed the Wnt/β‑catenin signaling pathway in OS. Invivo assays displayed the inhibitory role of the silencing of KCNQ1OT1 in OS tumor growth. As regards the underlying mechanisms, KCNQ1OT1 could sponge miR‑3666, and its expression was negatively associated with that of miR‑3666 in OS tissues. Thereafter, Kruppel‑like factor 7 (KLF7), upregulated in OS tissues and cells, was discerned as a target gene of miR‑3666. Furthermore, KLF7 expression negatively correlated with miR‑3666 expression, whereas it positively correlated with KCNQ1OT1 expression. A rescue assay delineated that the overexpression of KLF7 counteracted the KCNQ1OT1 knockdown‑induced suppression of OS cell proliferation, migration, invasion and Wnt/β‑catenin signaling. Collectively, the present study demonstrates that KCNQ1OT1 facilitates OS progression and activates Wnt/β‑catenin signaling by targeting the miR‑3666/KLF7 axis.

Amplified morphogenetic and bone forming activity of amorphous versus crystalline calcium phosphate/polyphosphate

Amorphous Ca-phosphate (ACP) particles stabilized by inorganic polyphosphate (polyP) were prepared by co-precipitation of calcium and phosphate in the presence of polyP (15% [w/w]). These hybrid nanoparticles showed no signs of crystallinity according to X-ray diffraction analysis, in contrast to the particles obtained at a lower (5% [w/w]) polyP concentration or to hydroxyapatite. The ACP/15% polyP particles proved to be a suitable matrix for cell growth and attachment and showed pronounced osteoblastic and vasculogenic activity in vitro. They strongly stimulated mineralization of the human osteosarcoma cell line SaOS-2, as well as cell migration/microvascularization, as demonstrated in the scratch assay and the in vitro angiogenesis tube forming assay. The possible involvement of an ATP gradient, generated by polyP during tube formation of human umbilical vein endothelial cells, was confirmed by ATP-depletion experiments. In order to assess the morphogenetic activity of the hybrid particles in vivo, experiments in rabbits using the calvarial bone defect model were performed. The particles were encapsulated in poly(d,l-lactide-co-glycolide) microspheres. In contrast, to crystalline Ca-phosphate (containing only 5% [w/w] polyP) or to crystalline β-tricalcium phosphate, amorphous ACP/15% polyP particles caused pronounced osteoinductive activity already after a six-week healing period. The synthesis of new bone tissue was accompanied by an intense vascularization and an increased expression of mineralization/vascularization marker genes. The data show that amorphous polyP-stabilized ACP, which combines osteoinductive activity with the ability to act as a precursor of hydroxyapatite formation both in vitro and in vivo, is a promising material for bone regeneration.