miR-125b-5p inhibits proliferation and migration of osteosarcoma cells by negatively regulating RAB3D expression / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect.@*METHODS@#The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments.@*RESULTS@#The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05).@*CONCLUSION@#Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

MicroRNA-629-5p promotes osteosarcoma proliferation and migration by targeting caveolin 1

Osteosarcoma is a highly malignant tumor that occurs in the bone. Previous studies have shown that multiple microRNAs (miRNAs) regulate the development of osteosarcoma. This study aimed to explore the role of miR-629-5p and its target gene, caveolin 1 (CAV1), in osteosarcoma development. To analyze the expression of miR-629-5p and CAV1 mRNA in osteosarcoma tissues and cell lines, qRT-PCR analysis was performed. Dual-luciferase reporter experiments were subsequently performed to validate the relationship between CAV1 and miR-629-5p. CCK8 assay was used to measure osteosarcoma cell proliferation, and wound-healing assay was performed to study their migratory phenotype. Our findings revealed that miR-629-5p was overexpressed in osteosarcoma tissues and cells, and thereby enhanced cell proliferation and migration. Further, we validated that miR-629-5p targets CAV1 mRNA directly. CAV1 expression, which was negatively correlated with miR-629-5p expression, was found to be downregulated in osteosarcoma tissue samples. Moreover, our data showed that an increase in CAV1 level led to a decline in osteosarcoma cell proliferation and migration, which could be rescued by miR-629-5p upregulation. Overall, our study confirmed that miR-629-5p promoted osteosarcoma proliferation and migration by directly inhibiting CAV1.

Down-regulation of HCP5 inhibits cell proliferation, migration, and invasion through regulating EPHA7 by competitively binding miR-101 in osteosarcoma

Patients with osteosarcoma (OS) usually have poor overall survival because of frequent metastasis. Long non-coding RNAs (lncRNAs) have been reported to be associated with tumorigenesis and metastasis. In this study, we investigated the expression and roles of lncRNA human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) in OS, aiming to provide a novel molecular mechanism for OS. HCP5 was up-regulated both in OS tissues and cell lines and high expression of HCP5 was associated to low survival in OS patients. Down-regulation of HCP5 inhibited cell proliferation, migration, and invasion, suggesting its carcinogenic role in OS. miR-101 was targeted by HCP5 and its expression was decreased in OS. The inhibitor of miR-101 reversed the impact of HCP5 down-regulation on cell proliferation, apoptosis, and metastasis in OS. Ephrin receptor 7 (EPHA7) was proved to be a target of miR-101 and had ability to recover the effects of miR-101 inhibitor in OS. In conclusion, lncRNA HCP5 knockdown suppressed cell proliferation, migration, and invasion, and induced apoptosis through depleting the expression of EPHA7 by binding to miR-101, providing a potential therapeutic strategy of HCP5 in OS.

miRNA-296-5p functions as a potential tumor suppressor in human osteosarcoma by targeting SND1 / 中华医学杂志(英文版)

BACKGROUND@#The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.@*METHODS@#We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.@*RESULTS@#We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).@*CONCLUSION@#Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.

Estudio clínico y molecular en una familia con osteocondromatosis múltiple / Clinical and molecular study in a family with multiple osteochondromatosis

Resumen: Se presentan dos casos de una familia con diagnóstico de osteocondromatosis múltiple, el cual fue confirmado por estudio molecular con mutación sin sentido en heterocigosis c.1219C>T, (p.Gln407Stop) en el gen EXT1. En el primer caso, en un paciente se presentó deformidad de Madelung como hallazgo infrecuente y en el otro caso, condrosarcoma como complicación temida, resaltando la variación intrafamiliar, por lo que se recomienda la evaluación individual e interdisciplinaria. Además, ante una entidad genética debe brindarse el adecuado y oportuno asesoramiento genético familiar a todos sus integrantes.

Abstract: We present two cases of a family with the diagnosis of multiple osteochondromatosis, which was confirmed by molecular study with nonsense in heterozygosis mutation c.1219C>T, (p.Gln407Stop) in the EXT1 gene. In these cases, the Madelung deformity was presented in one patient as an uncommon finding and chondrosarcoma as a feared complication in the other case, highlighting intrafamilial variation, which is why individual and interdisciplinary evaluation is recommended. In addition, before a genetic entity should provide adequate and timely family genetic counseling to all its members.

Up-regulated expression of CD147 gene in malignant bone tumor and the possible induction mechanism during osteoclast formation

It is increasingly evident that the microenvironment of bone can influence cancer phenotype in many ways that favor growth in bone. CD147, a transmembrane protein of the immunoglobulin (Ig) superfamily, was identified independently in different species and has many designations across different species. However, expression levels of CD147 mRNA in bone cancer have not been described. In this study, we have used real-time fluorescence quantification (RT-PCR) to demonstrate CD147 expression in malignant bone cancer and benign bone tumor tissues. The results suggested that the expression of CD147 gene was significantly up-regulated in malignant bone cancer. Moreover, we found that over-expressed RANKL progressively enhanced osteoclast formation up to 48 h, which suggested that RANKL could promote the formation of osteoclast, indicating that both CD147 and RANKL play important roles in the formation of osteoclasts. Furthermore, the expressions of four osteoclast specific expression genes, including TRACP, MMP-2, MMP-9 and c-Src, were analyzed using RT-PCR. The results indicated that four osteoclast-specific expression genes were detectable in all osteoclast with different treatments. However, the highest expression level of these four osteoclast-specific expression genes appears in the CD147+ RANKL group and the lowest expression level of these four osteoclast-specific expression genes appears with si-RANKL treatment. Characterization of the role of CD147 in the development of tumors should lead to a better understanding of the changes occurring at the molecular level during the development and progression of primary human bone cancer.

Long Non-Coding RNA TUG1 Promotes Proliferation and Inhibits Apoptosis of Osteosarcoma Cells by Sponging miR-132-3p and Upregulating SOX4 Expression

PURPOSE: Long non-coding RNA taurine upregulated gene 1 (TUG1) is reported to be a vital regulator of the progression of various cancers. This study aimed to explore the exact roles and molecular mechanisms of TUG1 in osteosarcoma (OS) development. MATERIALS AND METHODS: Real-time quantitative PCR was applied to detect the expressions of TUG1 and microRNA-132-3p (miR-132-3p) in OS tissues and cells. Western blot was performed to measure protein levels of sex determining region Y-box 4 (SOX4). Cell viability was assessed using XTT assay. Cell apoptosis was evaluated using flow cytometry and caspase-3 activity detection assays. Bioinformatics analysis and luciferase reporter experiments were employed to confirm relationships among TUG1, miR-132-3p, and SOX4. RESULTS: TUG1 was highly expressed in human OS tissues, OS cell lines, and primary OS cells. TUG1 knockdown hindered proliferation and induced apoptosis in human OS cell lines and primary OS cells. Moreover, TUG1 inhibited miR-132-3p expression by direct interaction, and introduction of miR-132-3p inhibitor partly abrogated the effect of TUG1 knockdown on the proliferation and apoptosis of OS cells. Furthermore, SOX4 was validated as a target of miR-132-3p. Further functional analyses revealed that miR-132-3p inhibited proliferation and induced apoptosis of OS cells, while this effect was greatly abated following SOX4 overexpression. Moreover, TUG1 knockdown suppressed proliferation and promoted apoptosis by upregulating miR-132-3p and downregulating SOX4 in primary OS cells. CONCLUSION: TUG1 facilitated proliferation and suppressed apoptosis by regulating the miR-132-3p/SOX4 axis in human OS cell lines and primary OS cells. This finding provides a potential target for OS therapy.

Investigating ego modules and pathways in osteosarcoma by integrating the EgoNet algorithm and pathway analysis

Osteosarcoma (OS) is the most common primary bone malignancy, but current therapies are far from effective for all patients. A better understanding of the pathological mechanism of OS may help to achieve new treatments for this tumor. Hence, the objective of this study was to investigate ego modules and pathways in OS utilizing EgoNet algorithm and pathway-related analysis, and reveal pathological mechanisms underlying OS. The EgoNet algorithm comprises four steps: constructing background protein-protein interaction (PPI) network (PPIN) based on gene expression data and PPI data; extracting differential expression network (DEN) from the background PPIN; identifying ego genes according to topological features of genes in reweighted DEN; and collecting ego modules using module search by ego gene expansion. Consequently, we obtained 5 ego modules (Modules 2, 3, 4, 5, and 6) in total. After applying the permutation test, all presented statistical significance between OS and normal controls. Finally, pathway enrichment analysis combined with Reactome pathway database was performed to investigate pathways, and Fisher's exact test was conducted to capture ego pathways for OS. The ego pathway for Module 2 was CLEC7A/inflammasome pathway, while for Module 3 a tetrasaccharide linker sequence was required for glycosaminoglycan (GAG) synthesis, and for Module 6 was the Rho GTPase cycle. Interestingly, genes in Modules 4 and 5 were enriched in the same pathway, the 2-LTR circle formation. In conclusion, the ego modules and pathways might be potential biomarkers for OS therapeutic index, and give great insight of the molecular mechanism underlying this tumor.

Tumores ósseos benignos e lesões ósseas pseudotumorais: avanços e atualização diagnóstica / Benign bone tumors and pseudotumors bone lesions:diagnostic advances and upgrade

O diagnóstico de tumores ósseos benignos e lesões ósseas pseudotumorais apresentou grandes avanços nos últimos anos. Entre os métodos de imagens, a radiografia simples permanece como o método de mais especificidade. A ressonância magnética e a tomografia computadorizada são valiosas no estadiamento e no planejamento cirúrgico, permitindo melhor avaliação da extensão do tumor e de sua ressecabilidade. A utilização de contraste e métodos dinâmicos permite visualizar alterações funcionais. A tomografia computadorizada por emissão de fótons (SPECT) associada à tomografia computadorizada (TC) permite a fusão direta de dados morfológicos e funcionais, sendo promissores para o estudo de doenças ósseas. A biópsia cirúrgica aberta é o padrão-ouro para o diagnóstico histológico de tumores músculo-esqueléticos. As técnicas percutâneas com agulha orientada por fluoroscopia, ultrassonografia e TC tornaram-se difundidas e diminuíram a necessidade de biópsia aberta, mas com menos acurácia diagnóstica. No campo genético, a citometria de fluxo mostra grande valor na diferenciação entre lesões benignas e malignas e para estadiamento tumoral. A descoberta da origem genética da exostose múltipla hereditária e da displasia fibrosa abre novas perspectivas para o diagnóstico e tratamento dos tumores ósseos hereditários. A identificação de inúmeros novos marcadores tumorais, moleculares e celulares relacionados com o comportamento e a agressividade tumoral permitirá o refinamento dos estadiamentos, o prognóstico e a criação de novos protocolos de tratamento.

The diagnosis of benign bone tumors and pseudotumors bone lesions showed great advances last years. Among the imaging methods, plain radiography is still the most specific method. Magnetic resonance imaging and computed tomography are very important in the staging and surgical planning, thus it allows a better assessment of the extent of the tumor and its resectability. Use of contrast and dynamic methods allows visualizing functional variations. Computed tomography for photon emission (SPECT) in association with computed tomography (CT) allows the direct fusion of functional and morphological data, once they are promising for the study of bone diseases. Open surgical biopsy is the gold standard for the histological diagnosis of musculoskeletal tumors. Imaging-guided Injection techniques with fluoroscopy, ultrasound and CT have become widespread and decreased the need for open biopsy, but with less diagnostic accuracy. In genetics, flow cytometry shows great importance in differentiating between benign and malignant lesions and for tumor staging. Hereditary multiple exostosis and of fibrous dysplasia have genetic origin. This fact shows new perspectives for diagnosis and treatment of hereditary bone tumors. Identification of several new tumor markers, molecular and cellular related to tumor behavior and aggressiveness will allow the refinement of staging, prognosis and development of new treatment protocols.

A comparison between genetic portraits of normal osteoblasts and osteosarcoma cell lines.

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor occurring in young patients in the first two decades of life. Metastases are the cause of 90% of cancer deaths for patients with OS. OS of the jaw is rare and aggressive malignancy constitutes approximately 5-13% of all cases of skeletal OS. Chemotherapy plus surgery are the first choice for treatment. AIMS: Because OS cell lines (OCLs) should share a common pathway with primary OS and new drugs are screened in in vitro systems, new insight about the genetic profiling of OCLs is of paramount importance to a better understanding of the molecular mechanism of this rare tumor and detecting a potential target for specific therapy. MATERIALS AND METHODS: The SAOS2 and TE85 cell lines were analysed using DNA microarrays containing 19,000 genes. Several genes in which expression was significantly differentially expressed in OCLs vs. normal osteoblast (NO) were detected. RESULTS: The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) cell differentiation, (c) apoptosis, and (d) immunity. CONCLUSION: The reported data can be relevant to a better understanding of the biology of OS and as a model for comparing the effect of drugs used in OS treatment.

Classical osteoblastoma, atypical osteoblastoma, and osteosarcoma: a comparative study based on clinical, histological, and biological parameters

OBJECTIVE: To investigate the biological behavior of classical and atypical osteoblastomas in comparison to osteosarcomas. METHODS: Based on histological parameters, 30 osteoblastomas were subclassified as classical osteoblastomas (23/30) or atypical osteoblastoma (high cellularity, prominent blue osteoid, and epithelioid osteoblasts-7/30). Comparative immunohistochemical and clinical analysis was performed in 17 cases of patients with high-grade osteosarcoma. Formalin-fixed, paraffin-embedded archival tissue was immunostained for p53 and proliferation cell nuclear antigen. Tumors with positive p53 stain underwent molecular analyses for fragments of exon 10. RESULTS: The mean proliferating cell nuclear antigen indexes for classical osteoblastoma, atypical osteoblastoma, and osteosarcoma were 33 percent, 61 percent, and 79 percent, respectively. The atypical subgroup showed similar results to those of the osteosarcoma group (P < 0.001). p53 protein was detected in 4 (13 percent) osteoblastomas (3 of these were atypical osteoblastoma), and 4 osteosarcomas (23 percent) also showed p53 positivity. DNA mutation performed in p53-positive cases was confirmed in exon 10 in 2 atypical osteoblastomas (2/3), 1 classical osteoblastoma (1/1), and 1 osteosarcoma (1/4). Disease recurrence was correlated with p53 expression (P = 0.009), atypical subtype (P = 0.031), spiculated blue bone on histology (P = 0.018), and proliferatingcell nuclear antigen labeling index > 40 (P = 0.015). CONCLUSION: These results validate atypical osteoblastoma as an entity, with p53 and proliferation cell nuclear antigen immunoexpression closer to that of osteosarcoma than of classical osteoblastoma. Proliferating cell nuclear antigen labeling index and p53 may be useful predictors of recurrence.

OBJETIVOS: Investigar o comportamento biológico de osteoblastomas clássicos e atípicos comparados com osteossarcomas. MÉTODOS: Com base em parâmetros histológicos classificamos um grupo de 30 osteoblastomas nos subgrupos de osteoblastomas clássicos (23/30) e de osteoblastomas atípicos (que apresentam como característica grande celularidade, osteóide azul proeminente e osteoblastos epitelióide-7/30). Como efeito de comparação dos resultados imunohistoquímicos e análise clínica, avaliamos 17 pacientes com osteosarcoma de grau avançado. Os cortes histológicos com bloco de parafina fixado em formalina foram imunocorados para p53 e antígeno nuclear de célula em proliferação. Tumores com coloração positiva para p53 tiveram análise molecular para fragmentos do exon 10. RESULTADOS: O índice médio de antígeno nuclear de célula em proliferação para osteoblastoma clássico, osteoblastoma atípico e osteosarcoma foram de 33 por cento, 61 por cento e 79 por cento, respectivamente. O subgrupo atípico demonstrou resultados similares aos dos osteosarcomas (p<0,001). Foram detectadas proteína p53 em 4 (13 por cento) osteoblastomas; 3 desses foram osteoblastomas atípicos, sendo que 4 osteosarcomas (23 por cento) também demonstraram p53 positivo. A mutação do DNA nos casos positivos de p53 foi confirmada no exon 10 em dois osteoblastomas atípicos (2/3), um osteoblastoma clássico (1/1) e um osteosarcoma (1/4). A recorrência da doença foi correlacionada com a expressão do p53 (p=0,009), subtipo atípico (p=0,031), osso azul espiculado no resultado da histologia (p=0,018), e índice de marcação pelo antígeno nuclear de célula em proliferação > 40 (p=0,015). CONCLUSÃO: Esses resultados validam os osteoblastomas atípicos como entidade real, com imunoexpressão das proteínas p53 e antígeno nuclear de célula em proliferação mais perto do osteosarcoma do que do osteoblastoma clássico. O índice de marcação pelo antígeno nuclear de célula em proliferação e o p53 podem...

Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients

Osteosarcoma is the commonest type of primary malignant bone tumor, frequently found in adolescents at sites of rapid bone growth. Despite current management protocols, up to half of the patients succumb to this disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. Since phage display methodology allows the selection of human antibody fragments with potential use in clinical applications, we applied this procedure to construct a recombinant Fab (antigen binding fragment) library from patients with osteosarcoma. We used peripheral blood lymphocyte total RNA from 11 osteosarcoma patients and cloned recombinant Fab representing the micro, gamma and kappa chain antibody repertoires of these individuals. The resulting library was cloned in the pComb3X vector and attained 1.45 x 10(8) different functional forms. BstO I fingerprinting and DNA sequencing analysis of randomly selected clones revealed the diversity of the library, demonstrating that Fab harbors Vkappa chains from subgroups I to V, biased towards the A27 fragment, as normally reported for the human repertoire. Analysis of the VH repertoire revealed that our library has a slight bias towards the VH4 family, instead of the usually reported VH3. This is the first description of a phage display library from osteosarcoma patients. We believe these human Fab fragments will provide a valuable tool for the study of this neoplasia and could also contribute to improvements in the diagnosis of this disease.

Tumours and tumour-like lesions of bone. The UHWI experience. University Hospital of the West Indies

A review of all primary tumours and tumour-like lesions of bone diagnosed in patients who attended the University Hospital of the West Indies over a 10-year period revealed 136 cases comprising 69 benign and 54 malignant tumours, and 13 cases of tumour-like lesions. The prevalence and clinical characteristics of the various lesions are discussed and compared with the findings in other series. The results of this analysis provide demographic data useful in the differential diagnosis of bone lesions locally.

Li-fraumeni syndrome in pediatric patients with soft tissue sarcoma or osteosarcoma

Family history of cancer and features of the Li-Fraumeni syndrome (LFS) were investigated in 42 patients with soft tissue sarcoma or osteosarcoma in a pediatric hospital in Mexico City, and compared with 42 non-cancer children. Six subjects with cancer were found among 204 first-degree relatives of cancer patients while there were none among 183 first-degree relatives of non-cancer children. In three families, the proband had two affected relatives, and the type of neoplasia as well as the age of onset suggested the clinical diagnosis of LFS. Our results show that 7.1 percent of our pediatric patients with soft tissue sarcoma or osteosarcoma may belong to LFS families. The authors encourage pediatric and adult oncologist to pay more attention to the history of cancer in nuclear families for eventual hereditary cancer syndrome identification and cancer prevention

Enhanced expression of cathepsin L in metastatic bone tumors

Cathepsin L is a kind of cystein proteases which are known to facilitate the invasion and metastasis of tumor cells by degrading the components of basement membrane and extracellular matrix. This study was undertaken to investigate the expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L in six normal tissues, two osteosarcoma cell lines, MG-63 and Saos-2, six primary bone tumors and six metastatic bone tumors. In six normal tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary > kidney > esophagus. One of the two osteosarcoma cell lines established from the primary sites expressed a high level of cathepsin L mRNA. Out of six primary bone tumors, three (50%) expressed cathepsin L mRNA, while all (100%) of six metastatic bone tumors expressed the mRNA. These results demonstrating the higher frequency of expression of cathepsin L in metastatic bone tumors suggest that cathepsin L may participate in tumor invasion and metastasis.

Associaçäo do retinoblastoma ao osteossarcoma: relato de dois casos / Retinoblastoma associated with osteosarcoma: report of two cases

O retinoblastoma é o tumor ocular mais freqüente na infância, geralmente diagnosticado nos primeiros três anos de vida, podendo ser uni ou bilateral. Acredita-se que seja de etiologia gênica e atualmente, com o aumento na sobrevida destes pacientes, tem-se observado outros tumores primários associados, mais freqëntemente o osteossarcoma osteoblástico. Os autores relatam dois casos de osteossarcoma osteoblástico em crianças que foram submetidas anteriormente a enucleaçäo de olho bilateral e unilateral, respectivamente, por retinoblastoma, O objetivo deste trabalho é apresentar dois novos casos na literatura médica e descrever consideraçöes sobre a correlaçäo entre as duas entidades.