RESUMO
Human RAD17 acts as an activator of checkpoint signals in response to DNA damage. Here we evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of colorectal cancer (CRC) in relation to smoking and alcohol consumption habits in 212 CRC patients and 1,142 cancer-free controls in a case-control study conducted in Japan. The results showed that the hRAD17 Leu/Arg genotype compared to the Leu/Leu genotypes was significantly associated with the protective effect on CRC risk with the adjusted odds ratio (OR) of 0.68 [95% confidence interval (CI): 0.49-0.95, p=0.024], and the males with the Arg/Arg genotype had a greater risk of CRC compared to those with the Leu/Leu and Leu/Arg genotypes (OR=1.87, 95%CI 1.03-3.40, p=0.04). In stratified studies, the protective effect of the Leu/Arg genotype on CRC risk was markedly higher in the light smokers (< 20 pack years) (OR=0.61, 95%CI 0.40-0.94, p=0.024) and the rectal cancer patients (OR=0.49, 95%CI 0.31-0.78, p=0.003). The risk of the Arg/Arg genotype was associated with heavy smoking (≥ 20 pack-years) (OR=2.24, 95%CI 1.09-4.61, p=0.03). These findings suggest that the genetic variant of hRAD17 Leu546Arg polymorphism has a significant effect on CRC susceptibility in Japanese.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Genótipo , Fumar/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Códon , Neoplasias Colorretais/etiologia , Intervalos de Confiança , Dano ao DNA , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Razão de Chances , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Adulto JovemRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally repress the expression of target genes. Many miRNAs have been implicated in a number of diseases, including cancers. The miR-17-92 miRNA cluster is known as a body of oncogenic miRNAs, and has been shown to be overexpressed in several cancers, including lung cancer. Although the overexpression of miR-17-92 is clearly implicated in the development of lung cancer, only a few direct targets for the miR-17-92 cluster have been identified thus far. In this study, we examined miR-17-92 target profiles in SBC-3 small-cell lung cancer cells using a quantitative proteomic strategy to identify direct targets of the miR-17-92 cluster. By knocking down the expression of endogenous miR-19a, miR-20a and miR-92-1, which are contained in the cluster, 112 up-regulated proteins were detected and also identified as potential targets of these miRNAs. Among these candidate targets, we validated one direct target, RAB14. In conclusion, these findings suggest that proteomic approaches are valuable for identifying direct miRNA targets, and we were able to identify a novel direct target for the miR-92-1 using our proteomic strategy.
Assuntos
Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteoma/genética , Carcinoma de Pequenas Células do Pulmão/genéticaRESUMO
Transcription factor ERG (erythroblast transformation-specific (ETS)-related gene) is essential in endothelial differentiation and angiogenesis, in which microRNA (miR)-200b-3p targeting site is expected by miRNA target prediction database. miR-200b is known decreased in hepatocellular carcinoma (HCC), however, the functional relation between ERG and miR-200b-3p, originating from pre-miR-200b, in HCC angiogenesis remains unclear. We investigated whether hepatocyte-derived miR-200b-3p governs angiogenesis in HCC by targeting endothelial ERG. Levels of miR-200b-3p in HCC tissues were significantly lower than those in adjacent non-HCC tissues. Poorly differentiated HCC cell line expressed lower level of miR-200b-3p compared to well-differentiated HCC cell lines. The numbers of ERG-positive endothelial cells were higher in HCC tissues than in adjacent non-HCC tissues. There was a negative correlation between the number of ERG-positive cells and miR-200b-3p expression in HCC tissues. Culture supernatants of HCC cell lines with miR-200b-3p-overexpression reduced cell migration, proliferation and tube forming capacity in endothelial cells relative to the control, while those with miR-200b-3p-inhibition augmented the responses. Exosomes isolated from HCC culture supernatants with miR-200b-3p overexpression suppressed endothelial ERG expression. These results suggest that exosomal miR-200b-3p from hepatocytes suppresses endothelial ERG expression, and decreased miR-200b-3p in cancer cells promotes angiogenesis in HCC tissues by enhancing endothelial ERG expression.
Assuntos
Carcinoma Hepatocelular/metabolismo , Células Endoteliais/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Neovascularização Patológica/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Endoteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Regulador Transcricional ERG/metabolismoRESUMO
NINEIN serves an essential role in centrosome function as a microtubule organizing center, and in the reformation of the interphase centrosome architecture following mitosis. In the present study, the association between NINEIN Pro1111Ala (rs2236316), a missense single nucleotide polymorphism, and the risk of colorectal cancer (CRC), related to smoking and alcohol consumption habits in 200 patients with CRC and 1,141 cancer-free control participants were assessed in a case-control study performed in Japan. The results showed that the NINEIN Ala/Ala genotype compared with the Pro/Pro genotype was significantly more associated with an increased risk of CRC, and the males with the Ala/Ala genotype exhibited a significantly increased risk of CRC compared with those with Pro/Pro and Pro/Ala genotypes. Stratified analyses of the Ala/Ala genotype with CRC risk further showed an increased association in never/light drinkers (<23 g of ethanol/day), in male never/light drinkers and in male patients with rectal cancer. These findings suggest that the genetic variant of the NINEIN Pro1111Ala polymorphism has a significant effect on CRC susceptibility in the Japanese population.
RESUMO
It has been reported that the threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene is correlated with acquired resistance to gefitinib. We previously reported that there was some population that harbored the EGFR T790M mutation as a minor clone of tumor cells prior to drug treatment, may be causing resistance to gefitinib during treatment. This fact also suggests that the detection of the EGFR T790M mutation prior to treatment may predict the development of resistance. We also showed that pleural fluid is a useful specimen for detection of EGFR mutation using sensitive assays. In this study, we reported a female patient who was treated with gefitinib because an EGFR L858R mutation was found in her pleural fluid. Our patient showed partial response to gefitinib, but she had progressive disease only 4 months after the start of treatment. Furthermore, the EGFR T790M mutation was detected in the pleural fluid before gefitinib treatment by the mutant-enriched PCR assay. Our findings confirmed that the EGFR T790M mutation was occasionally present as a minor population in tumor cells before treatment and caused resistance after gefitinib administration. The detection of a small fraction of T790M-positive alleles may be useful to predict the clinical course of the gefitinib-treated non-small-cell lung cancer patients.
Assuntos
Adenocarcinoma/genética , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Mutação , Derrame Pleural Maligno/genética , Quinazolinas/uso terapêutico , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamento farmacológico , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Seguimentos , Gefitinibe , Humanos , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/tratamento farmacológico , Reação em Cadeia da Polimerase , Prognóstico , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: Mutations in the epidermal growth factor receptor (EGFR) gene have been reported to be present in non-small cell lung cancer (NSCLC) and related to the responsiveness of tumors to EGFR tyrosine kinase inhibitors, suggesting its usefulness as a biomarker. Because clinical samples contain tumor and normal cells or genes, a highly sensitive assay for detecting mutation is critical for clinical applications. EXPERIMENTAL DESIGN: The mutant-enriched PCR is a rapid and sensitive assay with selective restriction enzyme digestion. We developed the mutant-enriched PCR assay targeting exons 19 and 21 of EGFR and applied the developed assay to detect mutations in 108 cases of surgically resected specimens of NSCLCs, 18 samples of computed tomography (CT)-guided needle lung biopsies, and 20 samples of pleural fluid. In addition, results were then compared with those from direct sequencing and a nonenriched PCR assay. RESULTS: The mutant-enriched PCR that was proved to enrich one mutant of 2 x 10(3) normal genes detected mutations in 37 cases of 108 resected tumors, seven samples of CT-guided lung biopsies, and seven samples of pleural fluid. Among mutant cases, four resected tumors, two CT-guided lung biopsies, and two pleural fluid were identified as additional mutant cases by the mutant-enriched PCR, which were considered normal based on nonenriched assays. CONCLUSIONS: Our results indicate that EGFR mutations are readily detectable by mutant-enriched PCR in various clinical samples. Thus, mutant-enriched PCR may provide a valuable method of potentially detecting a small fraction of mutant genes in heterogeneous specimens, indicating its possible use in clinical application for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genes erbB-1/genética , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase/métodos , Biópsia , Carcinoma Pulmonar de Células não Pequenas/patologia , Análise Mutacional de DNA , Primers do DNA , Feminino , Humanos , Neoplasias Pulmonares/patologia , Mutação , Derrame Pleural , Sensibilidade e EspecificidadeRESUMO
The incidence of lung adenocarcinoma has been increasing recently in smokers. The molecular target therapy has been developed for lung adenocarcinoma patients harboring EGFR gene mutation. However, the treatment modalities for patients without mutation are currently limited. Thus, analysis of EGFR gene mutation status at early stage is important strategy to classify the patients for improving treatments and prognosis efficiently. This study aimed to identify microRNA (miRNA) signature in relation to mutation status in EGFR gene in early stage of lung adenocarcinoma male patients with smoking history. MiRNA profiles were assessed by microarray in paired plasma and tissue pooled from 10 EGFR wild type (EGFR-wt) and 10 EGFR mutated (EGFR-mut) patients. Expressions of selected miRNAs were verified further by real-time qRT-PCR in 83 plasma samples consisting of 55 EGFR-wt patients and 28 EGFR-mut patients and their correlation with clinicopathological parameters and EGFR gene mutation status were evaluated. We found that seven miRNAs (miR-16-5p, miR-23a-3p, miR-103a-3p, miR122-5p, miR-223-3p, miR-346 and miR-451a) were differentially expressed in stage I and stage I+II. Especially, miR-23a-3p was only miRNA shown higher expression in EGFR-wt patients than EGFR-mut patients. Thus, our findings could be useful non-invasive biomarkers to differentiate mutation status in EGFR gene in smoker lung adenocarcinoma male patients.
RESUMO
PURPOSE: Aims of the study are to narrow-down the hotspot region on 10q21 defined by previous genome-wide loss of heterozygosity (LOH) analysis in head and neck squamous cell carcinomas (HNSCC) and to define candidate tumor suppressor genes (TSG) concerned with 10q21. MATERIALS AND METHODS: LOH analysis was carried out with ten polymorphic microsatellite markers. Expression analysis was performed by semi-quantitative RT-PCR, and mutation analysis by PCR and direct sequencing. RESULTS: LOH analysis on 10q21 in 52 HNSCC indicated distinctive and frequent allelic loss at D10S589 (42%). Among flanking genes, we found the RHOBTB1 gene as a candidate TSG, since an intragenic marker demonstrated the highest LOH (44%). Expression analysis revealed down-regulation of RHOBTB1 mRNA in 37% of tumors. Interestingly, all the five tumors that showed decreased expression of RHOBTB1 were accompanied with LOH, supporting the haploinsufficiency and class 2 TSG characteristics of RHOBTB1. No pathogenic mutation of RHOBTB1 was found. Furthermore, another gene within the region, EGR2, was also taken under scope. LOH frequencies around the EGR2 gene were relatively low (23 and 33%). Albeit semi-quantitative expression analysis of EGR2 demonstrated downregulation in 45% of tumor samples, no relation was found between the expression levels and LOH status. CONCLUSION: Frequent allelic loss and decreased expression of RHOBTB1 suggested that this gene has a role in tumorigenesis of a subset of HNSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 10 , Proteína 2 de Resposta de Crescimento Precoce/genética , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/genética , Perda de Heterozigosidade , Sequência de Bases , Análise Mutacional de DNA , Regulação para Baixo , Éxons , Regulação Neoplásica da Expressão Gênica , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Polimorfismo Genético , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The molecular pathogenesis of osteosarcoma is very complicated and associated with chaotic abnormalities on many chromosomal arms. We analyzed 12 cases of osteosarcomas with comparative genomic hybridization (CGH) to identify chromosomal imbalances, and detected highly frequent chromosomal alterations in chromosome 6q, 8p, 10p and 10q. To define the narrow rearranged region on chromosome 6 with higher resolution, loss of heterozygosity (LOH) analysis was performed with 21 microsatellite markers. Out of 31 cases, 23 cases (74%) showed allelic loss at least with one marker on chromosome 6q. We identified two distinct commonly deleted regions on chromosome 6 using markers D6S1565 located at 6q16 and 6q23MS1 at 6q23. The expression analysis of genes located at the deleted region was performed, and the decreased mRNA expression of the CCNC gene, one of the regulators of cell cycle, was detected. Growth of osteosarcoma cell line was significantly suppressed after the CCNC cDNA transfection. Fine mapping of the deleted region containing a possible tumor suppressor gene and the transfection assay suggest that the CCNC is a candidate tumor suppressor gene.
Assuntos
Cromossomos Humanos Par 6 , Ciclinas/genética , Deleção de Genes , Perda de Heterozigosidade , Osteossarcoma/genética , Osteossarcoma/patologia , Linhagem Celular Tumoral , Ciclina C , DNA Complementar/genética , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Hibridização de Ácido Nucleico , Mapeamento Físico do Cromossomo , RNA Mensageiro/metabolismoRESUMO
Human RAD17, a human homolog of the Schizosaccharomyces pombe cell cycle checkpoint gene RAD17, plays a significant role in activating checkpoint signals in response to DNA damage. We evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of esophageal squamous cell carcinoma in relation to smoking and alcohol consumption history in 154 esophageal squamous cell carcinoma male patients and 695 cancer-free male controls by a case-control study conducted in Japan. The results showed that the hRAD17 Arg/Arg genotype compared to the Leu/Leu and Leu/Arg genotypes was significantly associated with the risk of the esophageal squamous cell carcinoma with an adjusted odds ratios of 2.22 (95% CI: 1.19-4.16 P=0.013). In stratified studies, the risk of esophageal squamous cell carcinoma was markedly higher in light drinkers (less than 23 g ethanol/day) with the Arg/Arg genotype than in heavy drinkers (excess of 23 g ethanol/day) with the Arg/Arg genotype (OR=2.83, 95% CI: 1.05-7.61, P=0.04). We concluded that the genetic variant of hRAD17 Leu546Arg polymorphism exerts a significant effect on esophageal squamous cell carcinoma risk among Japanese men.
RESUMO
Loss of heterozygosity (LOH) has been frequently detected at chromosome 7q31 region in human head and neck squamous cell carcinomas (HNSCC) and many other cancers, suggesting the existence of tumor suppressor genes (TSG). We analysed LOH at 7q31 region in 49 HNSCC by using six polymorphic microsatellite markers and found allelic deletion in 48% (22/46) of the informative cases. We detected two preferentially deleted regions, one is around D7S643 and the other around D7S486. When we redefined the map of 7q31 region according to the contiguous sequences, a recently identified gene, ING3, was found in the proximity of D7S643. ING3 protein harbors the PHD domain highly homologous among ING family proteins, in which we previously found mutations in a related gene, ING1. As only one missense mutation of the ING3 gene was found in HNSCC, we examined the expression level. Reverse-transcription-PCR analysis demonstrated decreased or no expression of ING3 mRNA in 50% of primary tumors as compared with that of matched normal samples. Especially, about 63% of tongue and larynx tumors showed the decrease and a tendency of higher mortality was observed in cases with decreased ING3 expression. All these findings suggest a possibility that the ING3 gene functions as a TSG in a subset of HNSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 7/genética , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/genética , Proteínas/genética , Sequência de Aminoácidos , Northern Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Deleção Cromossômica , Mapeamento Cromossômico , Primers do DNA/química , DNA de Neoplasias/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Homeodomínio , Humanos , Perda de Heterozigosidade , Luciferases/metabolismo , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas , Proteínas/metabolismo , RNA/metabolismo , RNA Neoplásico/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transativadores , Proteínas Supressoras de TumorRESUMO
Micro RNAs (miRNAs) regulate the expression of target genes posttranscriptionally by pairing incompletely with mRNA in a sequence-specific manner. About 30% of human genes are regulated by miRNAs, and a single miRNA is capable of reducing the production of hundreds of proteins by means of incomplete pairing upon miRNA-mRNA binding. Lately, evidence implicating miRNAs in the development of lung cancers has been emerging. In particular, miR-19a, which is highly expressed in malignant lung cancer cells, is considered the key miRNA for tumorigenesis. However, its direct targets remain underreported. In the present study, we focused on six potential miR-19a target genes selected by miRNA target prediction software. To evaluate these genes as direct miR-19a target genes, we performed luciferase, pull-down, and western blot assays. The luciferase activity of plasmids with each miR-19a-binding site was observed to decrease, while increased luciferase activity was observed in the presence of anti-miR-19a locked nucleic acid (LNA). The pull-down assay showed biotinylated miR-19a to bind to AGO2 protein and to four of six potential target mRNAs. Western blot analysis showed that the expression levels of the four genes changed depending on treatment with miR-19a mimic or anti-miR-19a-LNA. Finally, FOXP1, TP53INP1, TNFAIP3, and TUSC2 were identified as miR-19a targets. To examine the function of these four target genes in lung cancer cells, LK79 (which has high miR-19a expression) and A549 (which has low miR-19a expression) were used. The expression of the four target proteins was higher in A549 than in LK79 cells. The four miR-19a target cDNA expression vectors suppressed cell viability, colony formation, migration, and invasion of A549 and LK79 cells, but LK79 cells transfected with FOXP1 and TP53INP1 cDNAs showed no difference compared to the control cells in the invasion assay.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genéticaRESUMO
CDH13 (H-cadherin) is a member of the cadherin superfamily, which plays an important role in cell recognition and adhesion. We examined the expression and methylation status of the CDH13 gene in diffuse large B cell lymphomas (B-DLCLs). We found decreased expression of the CDH13 gene in all of 6 hematopoietic cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Promoter hyper-methylation of the gene was detected in all 6 cell lines and in 13 of 19 (68%) B-DLCL samples by methylation-specific PCR. Interestingly, the methylation frequency of the CDH13 gene was comparable to those of the tumor suppressor genes p15 (68%) and p16 (74%) detected in B-DLCLs. Sequencing of bisulfite-treated DNA revealed hyper-methylation of the CpG islands of the CDH13 promoter in B-DLCLs and the cell lines. Treatment with 5-aza-2'-deoxycytidine restored CDH13 gene expression in a cell line in which promoter hyper-methylation and impaired expression of the CDH13 gene were observed. Loss of heterozygosity (LOH) around the CDH13 gene on chromosome 16q24 was detected in 6 of 15 (40%) informative cases with microsatellite marker D16S507 and in 6 of 15 (40%) cases with D16S422 in B-DLCLs. In all of 4 B-DLCL cases which showed both promoter methylation and LOH at the two marker loci, expression of the CDH13 gene was significantly low. These results suggest that silencing of the CDH13 gene by aberrant promoter methylation and allelic deletion is associated with tumorigenesis in a subset of B-DLCL.
Assuntos
Azacitidina/análogos & derivados , Caderinas/genética , Metilação de DNA , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Regiões Promotoras Genéticas , Azacitidina/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG/genética , Decitabina , Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/metabolismo , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/metabolismo , Prognóstico , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Certain tumor suppressor genes (TSG) residing on human chromosome 10q are implicated in astrocytic tumors. We thoroughly examined loss of heterozygosity (LOH) on chromosome 10q in astrocytic tumors to determine the extent of deletion and their relation to prognostic variables of patients. We analyzed 63 astrocytic tumors, including 9 diffuse astrocytomas, 36 anaplastic astrocytomas, and 18 glioblastomas. DNAs from tumors and leukocytes were analyzed for LOH at 18 microsatellite loci by polymerase chain reaction using fluorescence-labeled primers. Then correlation between LOH and clinicopathological variables was examined statistically. Twenty-four (66.7%) anaplastic astrocytomas and 15 (83.3%) glioblastomas had at least one LOH on chromosome 10q. However, diffuse astrocytomas exhibited no LOH. Nineteen tumors (10 anaplastic astrocytomas and 9 glioblastomas) were believed to have a total loss of one chromosome 10. Analyses on 20 tumors with interstitial LOH revealed that most of the high LOH regions matched the location of known TSGs, while some novel LOH regions were found preferentially in anaplastic astrocytoma. The median survivals of the total, partial, and no loss groups were 10.1, 14.8, and 46.8 months, respectively, indicating a significant difference in the survivals of these groups (P=0.0289). Thus, analyzing chromosome 10q loss is helpful for diagnosing malignancy in astrocytic tumors and for predicting patients' survival. Our data also suggested that there are novel TSGs for anaplastic astrocytoma at 10q24 and 10q26.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Cromossomos Humanos Par 10/genética , Genes Supressores de Tumor/fisiologia , Glioblastoma/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA de Neoplasias/genética , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , PrognósticoRESUMO
Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2;q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.
Assuntos
Proliferação de Células , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/genética , Sarcoma Sinovial/genética , Sarcoma Sinovial/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/análise , Proteínas de Fusão Oncogênica/análise , Proteínas Repressoras/análiseRESUMO
The miR-17-92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.
Assuntos
Neoplasias da Mama/genética , MicroRNAs/metabolismo , Proteômica/métodos , Algoritmos , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios Enzimáticos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Humanos , Luciferases/metabolismo , Células MCF-7 , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Família Multigênica/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligonucleotídeos/farmacologia , RNA Longo não Codificante , Reprodutibilidade dos TestesRESUMO
PURPOSE: We investigated the miRNA profile in peripheral nerve tumors and clarified the involvement of miRNA in the development and progression of MPNST in comparison with neurofibroma (NF). In addition, we attempted to seek associations between the miRNA and their potential targets in MPNST. METHODS: Global miRNA expression profiling was investigated for clinical samples of 6 MPNSTs and 6 NFs. As detected by profiling analysis, the expressions of miR-21 in clinical samples of 12 MPNSTs, 11 NFs, and 5 normal nerves, and 3 MPNST cell lines were compared using quantitative real-time reverse transcription PCR. MPNST cell line (YST-1) was transfected with miR-21 inhibitor to study its effects on cell proliferation, caspase activity, and the expression of miR-21 targets. RESULTS: Analysis of miRNA expression profiles in MPNST and NF revealed significantly altered expression levels of nine miRNAs, one of those, miR-21, and its putative target, programmed cell death protein 4 (PDCD4), were selected for further studies. miR-21 expression level in MPNST was significantly higher than that in NF (P < 0.05). In MPNST cells, transfection of miR-21 inhibitor significantly increased caspase activity (P < 0.01), significantly suppressed cell growth (P < 0.05), and upregulated protein level of PDCD4, indicating that miR-21 inhibitor could induce cell apoptosis of MPNST cells. CONCLUSIONS: These results suggest that miR-21 plays an important role in MPNST tumorigenesis and progression through its target, PDCD4. MiR-21 and PDCD4 may be candidate novel therapeutic targets against the development or progression of MPNSTs.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Transformação Celular Neoplásica/genética , MicroRNAs/genética , Neoplasias de Bainha Neural/genética , Proteínas de Ligação a RNA/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Linhagem Celular Tumoral , Análise por Conglomerados , Progressão da Doença , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/patologia , Neurofibroma/genética , Neurofibroma/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50% of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 3'-untranslated region of EGFR mRNA. Two EGFR-TKI-sensitive cell lines (PC-9 and H3255) and two EGFR-TKI-resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7-expressing plasmid. After transfection of the miR-7-expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92%. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7-expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7-expressing plasmids against EGFR oncogene-addicted lung cancers including T790M resistance by liposomal delivery.
Assuntos
Adenocarcinoma/terapia , Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/terapia , MicroRNAs/genética , Plasmídeos/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Regiões 3' não Traduzidas , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Apoptose/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Lipossomos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Proteína Oncogênica v-akt/metabolismo , Plasmídeos/genética , Transdução de Sinais , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only 1 T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 x 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of 111 cases) than in early-stage tumors (1 of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.
Assuntos
Substituição de Aminoácidos , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Antineoplásicos/uso terapêutico , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metionina , Mutação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Quinazolinas/uso terapêutico , Treonina , Proteínas ras/genéticaRESUMO
Spinal epidural lipomatosis (SEL) in patients on steroid therapy may be explained by two hypotheses: (1) steroids induce SEL and (2) steroids cause the growth of a pre-existing SEL, especially in obese children. Steroid-induced SEL (SSEL) is rarely described in children, with only six cases reported elsewhere. However, we have already reported four nephrotic children with SSEL, including one child in another hospital, in addition to the six cases. We investigated the frequency of SSEL in 125 children with renal diseases treated with glucocorticoids in a single hospital over 16 years, and examined the risk factors in 62 patients with SSEL reported in the literature. When patients complained of symptoms at the early stage of SSEL, i.e., back pain or numbness, and patients were obese, we performed spinal magnetic resonance imaging (MRI). SSEL was detected in 5 of 125 children (4.0%). Of the 5 patients with SSEL, 3 had vertebral compression fractures, and all 5 patients were on methylprednisolone pulse therapy. Our study suggests that many more patients can be detected at the early stage of SSEL by performing spinal MRI for patients with early symptoms, obesity, and those who have received methylprednisolone pulse therapy.