Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.426
Filtrar
1.
Medicine (Baltimore) ; 99(35): e21902, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32871922

RESUMO

The function of miR-9 in osteosarcoma is not well-investigated and controversial. Therefore, we conducted meta-analysis to explore the role of miR-9 in osteosarcoma, and collected relevant TCGA data to further testify the result. In addition, bioinformatics analysis was conducted to investigate the mechanism and related pathways of miR-9-3p in osteosarcoma.Literature search was operated on databases up to February 19, 2020, including PubMed, Web of Science, Science Direct, Cochrane Central Register of Controlled Trials, and Wiley Online Library, China National Knowledge Infrastructure, China Biology Medicine disc, Chongqing VIP, and Wan Fang Data. The relation of miR-9 expression with survival outcome was estimated by hazard ratio (HRs) and 95% CIs. Meta-analysis was conducted on the Stata 12.0 (Stata Corporation, TX). To further assess the function of miR-9 in osteosarcoma, relevant data from the TCGA database was collected. Three databases, miRDB, miRPathDB 2.0, and Targetscan 7.2, were used for prediction of target genes. Genes present in these 3 databases were considered as predicted target genes of miR-9-3p. Venny 2.1 were used for intersection analysis. Subsequently, GO, KEGG, and PPI network analysis were conducted based on the overlapping target genes of miR-9-3p to explore the possible molecular mechanism in osteosarcoma.Meta-analysis shown that overexpression of miR-9 was associated with worse overall survival (OS) (HR = 4.180, 95% CI: 2.880-6.066, P < .001, I = 23.5%). Based on TCGA data, osteosarcoma patients with overexpression of miR-9-3p (HR = 1.603, 95% CI: 1.028-2.499, P = .037) and miR-9-5p (HR = 1.698, 95% CI: 1.133-2.545, P = .01) also suffered poor OS. In bioinformatics analysis, 2 significant and important pathways were enriched: Wnt signaling pathway from gene ontology analysis (gene ontology:0016055, P-adjust = .008); hippo signaling pathway from Kyoto Encyclopedia of Genes and Genomes analysis (P-adjust = .007). Moreover, network analysis relevant protein-protein interaction was visualized, revealing 117 nodes and 161 edges.High miR-9 expression was associated with poor prognosis. Based on bioinformatics analysis, this study enhanced the understanding of the mechanism and related pathways of miR-9 in osteosarcoma.


Assuntos
MicroRNAs/genética , Osteossarcoma/genética , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Prognóstico , Transdução de Sinais/genética
2.
Int J Nanomedicine ; 15: 5131-5146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764941

RESUMO

Background: Gene therapy is considered a novel way to treat osteosarcoma, and microRNAs are potential therapeutic targets for osteosarcoma. miR-214 has been found to promote osteosarcoma aggression and metastasis. Graphene oxide (GO) is widely used for gene delivery for the distinct physiochemical properties and minimal cytotoxicity. Methods: Polyethyleneimine (PEI)-functionalized GO complex was well-prepared and loaded with miR-214 inhibitor at different concentrations. The load efficacy was tested by gel retardation assay and the cy3-labeled fluorescence of cellular uptake. The experiments of wound healing, immunofluorescence staining, Western blot, qRT-PCR and immunohistochemical staining were performed to measure the inhibitory effect of the miR-214 inhibitor systematically released from the complexes against MG63, U2OS cells and xenograft tumors. Results: The systematic mechanistic elucidation of the efficient delivery of the miR-214 inhibitor by GO-PEI indicated that the inhibition of cellular miR-214 caused a decrease in osteosarcoma cell invasion and migration and an increase in apoptosis by targeting phosphatase and tensin homolog (PTEN). The synergistic combination of the GO-PEI-miR-214 inhibitor and CDDP chemotherapy showed significant cell death. In a xenograft mouse model, the GO-PEI-miR-214 inhibitor significantly inhibited tumor volume growth. Conclusion: This study indicates the potential of functionalized GO-PEI as a vehicle for miRNA inhibitor delivery to treat osteosarcoma with low toxicity and miR-214 can be a good target for osteosarcoma therapy.


Assuntos
Grafite/química , MicroRNAs/antagonistas & inibidores , Terapia de Alvo Molecular , Osteossarcoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Polietilenoimina/química , Polietilenoimina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Terapia Combinada , Humanos , Camundongos , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia
3.
Zhonghua Yi Xue Za Zhi ; 100(21): 1668-1675, 2020 Jun 02.
Artigo em Chinês | MEDLINE | ID: mdl-32486604

RESUMO

Objective: To study the effects of miR-16-5p on proliferation, migration and invasion of osteosarcoma cells and its mechanism. Methods: Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression of miR-16-5p and TSPAN15 in human normal osteoblasts hFOB 1.19 and osteosarcoma cells MG63, Saos2 and HOS. The miR-16-5p or si-TSPAN15 was transfected into MG63 cells to observe its role in cell proliferation, migration and invasion. Cell proliferation was measured with MTT assay, cell migration and invasion were examined by Transwell, and the protein expression of CyclinD1, matrix metalloproteinase 2 (MMP-2), MMP-9, tetraspanin 15 (TSPAN15), phospha-tidylinositol3-kinase(p-PI3K) and phospha-protein kinase B(p-AKT) were determined by using Western blotting. The starbase website prediction combined with dual luciferase gene reporter assay was performed to analyze the targeting relationship between miR-16-5p and TSPAN15. miR-16-5p and pcDNA-TSPAN1 were co-transfected to assess the effect of high expression of TSPAN15 on overexpression of miR-16-5p-induced proliferation, migration and invasion of MG63 cells. Data comparison between the two groups was performed by using t test. Results: Compared with hFOB 1.19 cells (1.00±0.12), the expression of miR-16-5p was significantly decreased in MG63, Saos2 and HOS cells (0.32±0.05, 0.40±0.04, 0.45±0.06, respectively)(F=156.204, P<0.05), and TSPAN15 mRNA and protein levels were greatly increased (F=71.718, 110.350, both P<0.05). Overexpression of miR-16-5p obviously reduced the expression of CyclinD1, MMP-2, MMP-9 protein, cell viability, cell migration and invasion (F=150.136,117.228, 154.971, 89.479, 98.373, 130.880, all P<0.05) in MG63 cells. Knockdown of TSPAN15 greatly reduced CyclinD1, MMP-2, MMP-9 protein levels, cell survival rate, cell migration, and invasion number (F=93.206, 107.030, 109.326, 115.625, 146.113, 139.300, all P<0.05). Overexpression of miR-16-5p markedly decreased the expression of p-PI3K and p-AKT protein in MG63 cells (F=156.755, 181.419, both P<0.05). miR-16-5p targeted to regulate the expression of TSPAN15. High expression of TSPAN15 partially reversed the inhibitory effect of miR-16-5p on TSPAN15, CyclinD1, MMP-2, MMP-9, p-PI3K, p-AKT protein expression, cell viability, cell migration number and invasion number in MG63 cells. Conclusion: miR-16-5p inhibits the proliferation, migration and invasion of osteosarcoma cells by targeting the TSPAN15 gene and regulating the PI3K/AKT signaling pathway.


Assuntos
Neoplasias Ósseas , MicroRNAs/genética , Osteossarcoma , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Invasividade Neoplásica , Osteossarcoma/genética , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Tetraspaninas
4.
Life Sci ; 256: 117968, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32544462

RESUMO

Osteosarcoma (OS) is the most common type of primary bone malignancy with high recurrence and metastasis. Peptidylarginine deiminase 4 (PADI4), as an important protein post-translational modification enzyme, has been identified as a potential regulator in the invasion and migration in several types of tumors. The role of PADI4 in osteosarcoma metastasis remains unknown. In this study, we revealed significant positive correlation between PADI4 and pulmonary metastasis of osteosarcoma. Wound-healing and transwell assay indicated that PADI4 induced invasion and migration of osteosarcoma cell in vitro while PADI4 inhibitor has repressive effect. PADI4 mutation with no deimination activity exhibited no significant effect on invasion and migration of osteosarcoma cells. Moreover, we evaluated the effect of PADI4 on expression of the markers of epithelial-mesenchymal transition and results showed that PADI4 promoted EMT while PADI4 inhibitor suppressed EMT in osteosarcoma cells. We also detected the expression of PADI4 and E-Cadherin in the tissues of osteosarcoma patients with or without pulmonary metastasis. Results showed positive relationship between the expression of PADI4 and osteosarcoma metastasis. In contrast, the expression of E-Cadherin exhibited negative correlation with PADI4 and osteosarcoma metastasis. Our research offered a novel link between PADI4 and osteosarcoma metastasis and demonstrated PADI4 as a promising target for treatment of osteosarcoma metastasis.


Assuntos
Movimento Celular , Regulação para Baixo , Transição Epitelial-Mesenquimal , Osteossarcoma/enzimologia , Osteossarcoma/patologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/secundário , Invasividade Neoplásica , Osteossarcoma/genética , Proteína-Arginina Desiminase do Tipo 4/genética
5.
Life Sci ; 256: 117967, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32553931

RESUMO

AIMS: Magnoflorine is an essential type of alkaloid and possesses anti-tumor activity in multiple cancers. Recent studies have demonstrated that magnoflorine plays tumor-suppressive roles in gastric and breast cancers. However, its role in osteosarcoma (OS) tumorigenesis is enigmatic. This study aimed to investigate the role and mechanism of magnoflorine in OS. MATERIALS AND METHODS: Two human OS cells (MG-63 and U-2 OS) were treated with different concentrations of magnoflorine. Cell viability and invasion were then detected by Cell Counting Kit-8 and Transwell assay, respectively. And the effects of magnoflorine on the epithelial-mesenchymal transition (EMT) and cisplatin sensitivity were also measured. To explore the potential mechanism, we assayed the influence of magnoflorine on the miR-410-3p/HMGB1/NF-κB signaling pathway. Additionally, rescue experiments were performed to further confirm the regulation mechanism of magnoflorine. KEY FINDINGS: Magnoflorine inhibited the viability, invasion, and EMT of OS cells in a dose-dependent manner. And it increased the sensitivity of OS cells to cisplatin. Magnoflorine significantly suppressed HMGB1 expression and NF-κB activation, but upregulated miR-410-3p level. Overexpression of HMGB1 promoted NF-κB activation and reversed the effects of magnoflorine on the viability, invasion, EMT and cisplatin sensitivity of OS cells. miR-410-3p mimic inhibited the EMT of OS cells, which was restored by HMGB1 upregulation. And miR-410-3p inhibitor abrogated the influence of magnoflorine on HMGB1 expression in OS cells. SIGNIFICANCE: Magnoflorine inhibited the malignant phenotypes and increased cisplatin sensitivity of OS cells via modulating miR-410-3p/HMGB1/NF-κB pathway. These results indicated that magnoflorine might be a novel drug for the treatment of OS.


Assuntos
Aporfinas/farmacologia , Cisplatino/farmacologia , Proteína HMGB1/metabolismo , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Osteossarcoma/patologia , Transdução de Sinais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Osteossarcoma/genética , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
6.
DNA Cell Biol ; 39(7): 1172-1180, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32584170

RESUMO

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.


Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Biologia Computacional , Terapia de Alvo Molecular , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Neoplasias Ósseas/diagnóstico , Bases de Dados Genéticas , Ontologia Genética , Humanos , Osteossarcoma/diagnóstico , Prognóstico , Transcriptoma
7.
Medicine (Baltimore) ; 99(26): e20486, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590731

RESUMO

BACKGROUND: Single nucleotide polymorphisms (SNPs) have been inconsistently associated with osteosarcoma (OS) risk. This meta-analysis aimed to synthesize relevant data on SNPs associated with OS. METHODS: Databases were searched to identify association studies of SNPs and OS published through January 2020 from the databases of PubMed, Web of Science, Embase, Cochrane Library, China National Knowledge Infrastructure, the Chinese Science and Technology Periodical Database, and Wan fang databases. Network meta-analysis and Thakkinstian algorithm were used to select the most appropriate genetic model, along with false positive report probability for noteworthy associations. The methodological quality of data was assessed based on the STrengthening the REporting of Genetic Association Studies statement Stata 14.0 will be used for systematic review and meta-analysis. RESULTS: This study will provide a high-quality evidence to find the SNP most associated with OS susceptibility and the best genetic model. CONCLUSIONS: This study will explore which SNP is most associated with OS susceptibility. REGISTRATION: INPLASY202040023.


Assuntos
Neoplasias Ósseas/genética , Osteossarcoma/genética , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Humanos , Metanálise em Rede , Projetos de Pesquisa , Medição de Risco , Revisões Sistemáticas como Assunto
8.
Zhonghua Zhong Liu Za Zhi ; 42(4): 325-330, 2020 Apr 23.
Artigo em Chinês | MEDLINE | ID: mdl-32375449

RESUMO

Objective: To study the effect of long non-coding RNA LINC-PINT on proliferation and apoptosis of osteosarcoma cells. Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of LINC-PINT and miR-524-5p in normal osteoblast hFOB and human osteosarcoma cell lines HOS, MG63 and SAOS2 cells. The pcDNA plasmid, pcDNA-LINC-PINT plasmid, negative control siRNA (si-NC), si-LINC-PINT, negative control mimics (miR-NC), miR-524-5p mimics (miR-524-5p), pcDNA-LINC-PINT combined with miR-NC, pcDNA-LINC-PINT combined with miR-524-5p were transfected into HOS cells with liposome, respectively. The protein expressions of PCNA and cleaved-caspase-3 in the cells were detected by western blot. Cell proliferation ability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The apoptosis was detected by flow cytometry. The transcriptional activity was detected by double luciferase reporter assay. Results: Compared with normal osteoblast hFOB cell (1.00±0.08 vs 1.00±0.06), the expressions of LINC-PINT were down-regulated (0.18±0.01; 0.33±0.01; 0.42±0.01), while the expressions of miR-524-5p were up-regulated (2.65±0.23; 1.68±0.14; 1.51±0.13) in human osteosarcoma cell lines HOS, MG63 and SAOS2 cells, respectively. Overexpression of LINC-PINT significantly inhibited the proliferation (0.41±0.05 vs. 0.62±0.05 for 48 h; 0.57±0.05 vs. 1.06±0.09 for 72 h, both P<0.05) while promoted the apoptosis (25.28±2.15 vs. 9.01±0.17, P<0.01) of HOS cells. Knockdown of LINC-PINT or overexpression of miR-524-5p can significantly promote the proliferation and inhibit apoptosis of HOS cells. Moreover, miR-524-5p inhibited the fluorescence activity of wild-type LINC-PINT (0.31±0.03) in HOS cells when comparred with miR-NC (1.00±0.03) and was negatively regulated by LINC-PINT. Overexpression of miR-524-5p reversed the proliferation inhibition and apoptosis-promotion effects of LINC-PINT in HOS cells. Conclusions: Long non-coding RNA LINC-PINT can inhibit the proliferation and promote apoptosis of osteosarcoma cells through targeting miR-524-5p, which will provide a new target for the treatment of osteosarcoma.


Assuntos
Neoplasias Ósseas/genética , MicroRNAs/genética , Osteossarcoma/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Apoptose , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Osteossarcoma/patologia
9.
Yonsei Med J ; 61(5): 359-370, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32390359

RESUMO

PURPOSE: Osteosarcoma (OS) is the most common primary bone tumor, with high morbidity in infants and adolescents. Long noncoding RNA LINC00313 has been found to modulate papillary thyroid cancer tumorigenesis and to be dysregulate in lung cancer. However, the role of LINC00313 in OS has not yet been addressed. MATERIALS AND METHODS: We evaluated mRNA and protein expression using real-time quantitative PCR and Western blotting. Cell proliferation was evaluated using MTT; apoptosis and autophagy were assessed with flow cytometry, Western blotting, and/or GFP-LC3 assay. Transwell assay was conducted to measure cell migration and invasion. Potential target sites for LINC00313 and miR-342-3p were predicted with starBase v.2.0 and TargetScan Human, and verified using luciferase reporter assay, RNA immunoprecipitation, and RNA pull-down assay. In vivo, xenogeneic tumors were induced with U2OS and MG-63 cells, separately. RESULTS: LINC00313 was upregulated and miR-342-3p was downregulated in OS tissues and cells. High expression of LINC00313 was associated with shorter overall survival. FOSL2 downregulation and miR-342-3p overexpression suppressed cell proliferation and migratory and invasive abilities while promoting apoptosis and autophagy, all of which were consistent with the effects of LINC00313 knockdown. miR-342-3p, sponged by LINC00313, inversely modulated FOSL2 by targeting MG-63 cells, and FOSL2 expression was positively controlled by LINC00313. LINC00313 knockdown suppressed tumor growth in vivo. CONCLUSION: LINC00313 is upregulated in OS, and LINC00313 knockdown plays a vital anti-tumor role in OS cell progression through a miR-342-3p/FOSL2 axis. Our study suggests that LINC00313 may be a novel, promising biomarker for diagnosis and prognosis of OS.


Assuntos
Carcinogênese/patologia , Antígeno 2 Relacionado a Fos/genética , Técnicas de Silenciamento de Genes , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Longo não Codificante/genética , Regulação para Cima/genética , Adulto , Sequência de Bases , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Antígeno 2 Relacionado a Fos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Metástase Neoplásica , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
10.
J Biol Regul Homeost Agents ; 34(2): 517-524, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32450677

RESUMO

Small nucleolar RNA host genes (SNHGs) as a subset of long noncoding RNAs (lncRNAs) have critical roles in the pathogenesis of multiple malignancies, however, the role and molecular mechanisms of lncRNA SNHG8 in osteosarcoma (OS) remain unclear. In the present study, the correlation of SNHG8 or miR-542-3p with clinicopathological elements and prognosis in OS patents was estimated by TCGA cohort. Cell viability and invasion were assessed by MTT and Transwell assays. The interplay between SNHG8 and miR-542-3p was affirmed by a luciferase report assay. The effects of SNHG8 on miR-542-3p expression were examined in MG-63 and SW-1353 cells by qRT-PCR analysis. The results showed that incremental expression of SNHG8 or reduced expression of miR-542-3p was related to poor survival and tumor recurrence in OS patients. Overexpressing SNHG8 accelerated the growth and invasion of MG-63 cells, but silencing SNHG8 harbored an opposite effect in SW-1353 cells. Additionally, SNHG8 could negatively regulate miR-542-3p expression and bind with miR-542-3p, which attenuated SNHG8 induced cell proliferation. Taken together, these findings indicate that lncRNA SNHG8 promotes the proliferation of OS cells by downregulating miR-542-3p.


Assuntos
Neoplasias Ósseas/patologia , MicroRNAs/genética , Osteossarcoma/patologia , RNA Longo não Codificante/genética , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Recidiva Local de Neoplasia , Osteossarcoma/genética
11.
J Cancer Res Clin Oncol ; 146(7): 1693-1700, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32333142

RESUMO

PURPOSE: Osteosarcoma is the most common bone tumor, mainly affecting adolescents and young adults, and metastatic disease has poor outcomes with a dismal overall survival. Currently, chemotherapy is the standard of care with limited results, finding that new therapies could improve these outcomes. Preclinical and clinical studies have suggested a possible important role of ErbB pathway aberrations in osteosarcoma etiology. The present study shows the effect of afatinib, an irreversible ErbB family blocker in osteosarcoma cell lines. METHODS: Within a panel of human osteosarcoma cell lines, we addressed cell viability assay using afatinib at increasing concentrations. Motility was measured in wound-healing assays and invasion capacity was assessed in Transwell chamber assays. Finally, to monitor ErbB pathway modulation by afatinib and related compounds, we used Western blot analyses. RESULTS: Cell viability inhibition, as well as a reduction of motility and migration of osteosarcoma cell line were observed after treatment with afatinib. Likewise, in the HOS cell line, afatinib decreased phosphorylation of key components in the ErbB signaling pathway. CONCLUSIONS: Afatinib shows relevant antitumor effect in several osteosarcoma cell lines, as it causes a significant impact on cell viability, motility, and migration with a significant decrease in the activation of ErbB pathway activity.


Assuntos
Afatinib/farmacologia , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Arch Biochem Biophys ; 686: 108351, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32240636

RESUMO

Transforming growth factor beta regulator 4 (TBRG4) is a novel regulator in tumorigenic progression of several tumors. However, so far, the expression and functions of TBRG4 in osteosarcoma are unknown. The aim of this study was to investigate the potential biological functions of TBRG4 in osteosarcoma. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of TBRG4 in osteosarcoma tissues and cell lines. The levels of TBRG4 protein in osteosarcoma tissues were assessed by immunohistochemistry. Lentivirus-mediated short hairpin (sh) RNA was employed to knock down TBRG4 in osteosarcoma cells, and the expressions of TBRG4 mRNA and protein were determined by qRT-PCR and Western blot assay, respectively. Subsequently, the proliferation, clonogenic ability, apoptosis and invasion of osteosarcoma cells were measured using high content screening analysis and CCK8 assay, tumor sphere formation assay, flow cytometry and Transwell invasion assays, respectively. Furthermore, the osteosarcoma cells growth and metastasis in vivo were detected, and the effect of TBRG4 on the transforming growth factor ß1 (TGF-ß1) and PI3K/AKT signaling pathway was explored by qRT-PCR and Western blot assay, respectively. The results showed the levels of TBRG4 were overexpressed in osteosarcoma tissues and cell lines, confirming that the high TBRG4 expression was related to advanced tumor stages, large tumor size, and lymph node metastasis. Functional assays showed knockdown of TBRG4 could inhibit proliferation, invasion and induce apoptosis of osteosarcoma cells in vitro, and could also suppress osteosarcoma growth and metastasis in vivo. By examining the expression levels of TGF-ß1, p-PI3K, PI3K, p-AKT and AKT, it showed that the suppression of TBRG4 would reduce TGF-ß1 expression and inactivate the PI3K/AKT signaling pathway. These results showed for the first time that TBRG4 knockdown could suppress osteosarcoma progression, suggesting TBRG4 might be a promising therapeutic target for osteosarcoma treatment.


Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas Mitocondriais/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lentivirus , Camundongos Nus , Proteínas Mitocondriais/genética , Invasividade Neoplásica , Osteossarcoma/terapia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Hum Cell ; 33(3): 810-818, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32274658

RESUMO

Circular RNAs (circRNAs) exert pivotal effects on regulating the progression of osteosarcoma (OS). It was found through microarray analysis that circ-0002052 is abnormally expressed in OS, but the role of circ-0002052 in OS remains obscure. The results of this research manifested that relative to that in non-tumor controls, circ-0002052 level was raised in OS tissues. Up-regulated circ-0002052 was associated with advanced stage, tumor size, and metastasis. Additionally, circ-0002052 elevation indicated a low survival rate in OS patients and silencing of circ-0002052 suppressed proliferation, migration, and invasion of OS cells. It was proved that circ-0002052 sponged miR-382 and stimulated STX6 expression, thus activating Wnt/ß-catenin. The function of circ-0002052 reduction in OS cells was effectively reversed by miR-382 suppression. To sum up, it can be concluded that circ-0002052, functioning as a sponge for miR-382, enhances the activation of Wnt/ß-catenin mediated by STX6 to stimulate the progression of OS, and circ-0002052 may be an underlying treatment target and a biomarker for prognosis of osteosarcoma.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Circular/fisiologia , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Osteossarcoma/diagnóstico , Osteossarcoma/terapia , RNA Circular/genética , RNA Circular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
14.
Med Sci Monit ; 26: e920826, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193367

RESUMO

BACKGROUND This study aimed to investigate the role of gene mutation site distribution, biological function, pathway enrichment, and gene association analysis in the occurrence, development, and migration of osteosarcoma. MATERIAL AND METHODS Somatic mutation screening was performed using the whole-exome sequencing of osteosarcoma samples, and the distribution of mutations was demonstrated by Circos diagrams. Metascape was used to analyze the GO and KEGG signal pathway enrichment of the genes harboring protein coding alterations, and GeneMANIA was used to analyze the interaction of mutated genes. RESULTS The results showed that the protein coding alterations were found throughout the whole genome in 3 osteosarcoma samples. A large number of identical or related biological processes and pathways were found in osteosarcoma samples. The GeneMANIA analysis of the 10 mutations shared by 3 samples showed that the target gene minichromosome maintenance complex component 4 (MCM4) and 3 lateral genes were most functional, and were all related to DNA replication. The analysis of GO and KEGG signal pathway enrichment showed that the mutated genes were involved mainly in tumor-related metabolic pathways. Three mutated genes were involved in the cell process, and 2 mutated genes were involved in the metabolic process. Known driver gene mutations were also observed in the samples. CONCLUSIONS The gene analysis confirmed that patients with osteosarcoma had a wide range of common gene mutations related to each other, which are involved in tumor-related metabolic pathways. These findings provide a basis for further gene-targeted therapy and pathway research.


Assuntos
Neoplasias Ósseas/genética , Mutação , Osteossarcoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Transdução de Sinais , Sequenciamento Completo do Exoma
15.
Nat Commun ; 11(1): 1141, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111827

RESUMO

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.


Assuntos
Caseína Quinase Ialfa/metabolismo , Ligases/antagonistas & inibidores , Ligases/metabolismo , Osteossarcoma/patologia , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caseína Quinase Ialfa/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligases/genética , Camundongos , Mutação , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas do Grupo Polycomb/genética , Regiões Promotoras Genéticas , Compostos de Pirvínio/farmacologia , Compostos de Pirvínio/uso terapêutico , Análise de Sobrevida , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
16.
Int J Clin Oncol ; 25(6): 1195-1205, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32215805

RESUMO

OBJECTIVE: The aim of this study was to investigate the efficacy and safety of Apatinib mesylate in the treatment of metastatic osteosarcoma patients who progressed after standard therapy and the VEGFR2 gene polymorphism analysis. METHODS: Designed as a retrospective study, a total of 105 metastatic osteosarcoma patients who progressed after standard therapy were included in this study. The metastatic osteosarcoma patients received 500-750 mg Apatinib mesylate according to body surface area until disease progression or unacceptable toxicity with 28 days one cycle. Overall response was evaluated after two cycles Apatinib treatment, then progression-free survival (PFS) and overall survival (OS) were evaluated, and safety data were recorded. Additionally. peripheral blood and peripheral blood mononuclear cell (PBMC) specimens in the osteosarcoma patients were collected for the genotyping of VEGFR2 genetic variation and mRNA expression, respectively. Analysis on the association between genotype and baseline characteristics and VEGFR2 gene mRNA expression was analyzed. The univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, and multivariate analysis was adjusted by Cox regression analysis. RESULTS: The objective response rate (ORR) of the 105 metastatic osteosarcoma patients was 37.14%, disease control rate (DCR) was 77.14%, median PFS was 4.1 months, and median OS was 9.0 months. Regarding the VEGFR2 gene polymorphisms analysis, only - 906 T > C was of clinical significance. The prevalence of - 906 T > C in VEGFR2 among the study population was as follows: TT genotype 62 cases (59.05%), TC genotype 36 cases (34.29%) and CC genotype 7 cases (6.66%), minor allele frequency of - 906 T > C was 0.24. Compared with patients with TC/CC genotype, patients with TT genotype showed longer median PFS (5.0 versus 3.1 months, P = 0.011) and median OS (9.8 versus 7.6 months, P = 0.032). There was no correlation between the polymorphism and adverse reactions. Additionally, the mRNA expression in 69 randomly selected sample indicated that the mRNA expression of VEGFR2 of the patients with CC/TC genotypes were significantly higher than those of the TT genotype patients (P < 0.001). CONCLUSION: Apatinib was safe and effective in the treatment of metastatic osteosarcoma patients who progressed after standard therapy. The clinical outcomes of Apatinib may be influenced by the polymorphism - 906 T > C of VEGFR2 through mediating the mRNA expression of VEGFR2.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Polimorfismo Genético , Piridinas/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Adolescente , Adulto , Neoplasias Ósseas/genética , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/fisiologia , Masculino , Pessoa de Meia-Idade , Osteossarcoma/genética , Osteossarcoma/mortalidade , Osteossarcoma/patologia , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
17.
Neoplasma ; 67(3): 519-527, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32122144

RESUMO

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with different types of cancer. A novel paradigm in tumor biology suggests that the initiation and progression of osteosarcoma (OS) are driven by osteosarcoma stem cell-like cells (OSCs), but the role and underlying mechanisms of KLF8 in OSCs are poorly elucidated. In this study, an obviously increased level of KLF8 is shown in 9 out of 10 primary OS tissues and is associated with the poor progression-free interval. Significantly, KLF8 expression in CD133+ OSCs is higher than that in CD133- counterparts. By knocking down KLF8 in CD133+ OSCs, we show that si-KLF8-OSCs can hardly form compact spheres. In the meantime, infection with si-KLF8 in CD133+ OSCs results in the downregulation of OCT4 and SOX2; increased adriamycin (ADM) sensitivity; and decreased tumorigenic potential in vivo. Mechanisms study demonstrates that KLF8 directly binds the miR-429 promoter region and regulates its expression transcriptionally. Furthermore, we indicate that miR-429 directly targets SOX2 to mediate cancer stem cell-like features in CD133+ OSCs. In the clinic, miR-429 levels are negatively associated with KLF8 levels in OS, suggesting that an elevated KLF8/miR-429 ratio may have clinical value as a predictive biomarker. In conclusion, targeting the KLF8-miR-429-SOX2 signaling pathway may provide an effective therapeutic approach to suppress the initiation and progression of OS.


Assuntos
Neoplasias Ósseas/patologia , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/citologia , Osteossarcoma/patologia , Fatores de Transcrição SOXB1/genética , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Osteossarcoma/genética , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais
18.
Technol Cancer Res Treat ; 19: 1533033820909125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32129151

RESUMO

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


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , MicroRNAs/genética , Osteossarcoma/patologia , Adulto , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Transição Epitelial-Mesenquimal , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Masculino , MicroRNAs/metabolismo , Invasividade Neoplásica , Estadiamento de Neoplasias , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Adulto Jovem
19.
Nat Commun ; 11(1): 1008, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081846

RESUMO

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.


Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Osteossarcoma/genética , Osteossarcoma/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/patologia , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Fenômenos Imunogenéticos , Masculino , Pessoa de Meia-Idade , Mutação , Osteossarcoma/secundário , RNA-Seq , Receptores de Antígenos de Linfócitos T/genética , Sequenciamento Completo do Genoma , Adulto Jovem
20.
J Vis Exp ; (156)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32065171

RESUMO

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4-5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.


Assuntos
Neoplasias Ósseas/genética , Metilação de DNA , Vesículas Extracelulares/genética , Elementos Nucleotídeos Longos e Dispersos , Células-Tronco Mesenquimais/metabolismo , Osteossarcoma/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Meios de Cultivo Condicionados/metabolismo , DNA/genética , Epigênese Genética , Vesículas Extracelulares/metabolismo , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA