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1.
Clin Lung Cancer ; 2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31761448

RESUMO

BACKGROUND: Neurotrophin receptor kinase (NTRK) gene fusions (NTRK+) are rare but actionable oncogenic drivers present in a wide variety of solid tumors. However, the clinicopathologic characteristics of NTRK1 fusion-positive non-small-cell lung cancer are largely unknown. MATERIALS AND METHODS: Lung cancer tissue specimens and/or circulating cell-free DNA from patients with lung cancer who had undergone molecular profiling at a Clinical Laboratory Improvement Amendments (CLIA)-certified genomics laboratory in China were retrospectively reviewed. The laboratory performed NTRK1 fusion detection using hybridization-based targeted next-generation sequencing. The patients' clinical characteristics and treatment history were retrieved from the database for further evaluation. RESULTS: A total of 21,155 Chinese lung cancer cases had undergone molecular profiling from April 2016 to March 2019, including 13,630 adenocarcinoma cases. Of these cases, 12 were positive for NTRK1 fusion, including 10 cases of adenocarcinoma (0.073%), 1 primary sarcomatoid carcinoma, and 1 with an unknown histologic classification. Seven fusion partners (CD74, interferon regulatory factor 2 binding protein 2 [IRF2BP2], lamin A/C [LMNA], PHD finger protein 20 [PHF20], sequestosome 1 [SQSTM1], tropomyosin 3 [TPM3], TPR) were identified. Additionally, 1 unique rearrangement occurred upstream of the transcription start site of BCL9 fused to exon 12 of NTRK1 (intragenic region, BCL9-NTRK1). Of the 12 cases of NTRK1+ lung cancer, 6 had had concurrent activating EGFR mutations and/or had received previous treatment with EGFR tyrosine kinase inhibitors (TKIs), with 2 having concurrent EGFR T790M and 1 additional EGFR C797S. CONCLUSIONS: NTRK1+ lung cancer cases are extremely rare with multiple fusion partners. Additionally, emergence of NTRK1+ fusion might act as a resistance mechanism to EGFR TKIs. When performing comprehensive analysis of acquired resistance to EGFR TKIs, the ability to detect NTRK1 fusions will be important.

2.
J Cancer ; 10(21): 5070-5081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31602259

RESUMO

Hepatocyte growth factor (HGF) expression is repressed in normal differentiated lung epithelial cells, but its expression is aberrantly upregulated in non-small cell lung cancer (NSCLC) and acts as a poor prognostic factor. The underlying molecular mechanisms of aberrant HGF expression are unclear. In this study, a novel differential methylation region located in the HGF promoter was identified, which was associated with aberrant HGF expression in NSCLC. The correlations of HGF promoter methylation detected by methylation specific PCR and HGF expression detected by immunohistochemistry with clinical outcomes were assessed in NSCLC patients. DNA methylation of the HGF promoter was correlated with the activation of HGF expression, which induced epithelial-mesenchymal transition, cell migration and invasion. According to the clinical correlation analysis in 63 NSCLC patients, those with high methylation were more likely to have stages III and IV (51.6% vs. 25.0%, P<0.05) and metastasis (57.5% vs. 16.7%, P<0.05) than patients with low methylation. In addition, compared with the protein marker of HGF expression, the DNA methylation marker of the HGF promoter had higher specificity for prognostic analysis of metastases in NSCLC. Our study indicated the regulatory mechanisms related to DNA methylation of the HGF promoter for HGF expression in NSCLC epithelial cells, and suggested that the DNA methylation signature of the HGF promoter could potentially be employed as a biomarker to improve the prognostic accuracy of NSCLC.

3.
Biol Pharm Bull ; 42(3): 432-441, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828075

RESUMO

Moutan Cortex charcoal has been used to ameliorate blood heat symptoms and treat pathologic hemorrhage down the ages. Although well known as an agent with the effect of astringency and hemostasis, its active ingredients and action mechanism remain unclear. In the present study, molecular docking technology was employed to screen the potential hemostatic compounds in Moutan Cortex charcoal and their target proteins. Protein-protein-interaction (PPI) analysis was performed to explain the functions and enrichment pathways of the target proteins. The results showed that a total of 25 compounds were estimated as active constituents targeting multiple proteins related to hemostatic diseases, including 5 proteins (SERPINC1, FVIII, FX, FII and FXII) that were considered as the key targets. Then the drug-target (D-T) network was constructed to analyze the underlying hemostatic mechanism of Moutan Cortex charcoal, followed by a hierarchical cluster analysis (HCA) for compounds clustering, and a coagulation screening test for compound verification on their coagulation activities, with the results indicating that M15 (5-Tetradecenoic acid) and M31 (1-Monolinolein) might be the key compounds contributing to the hemostasis effect of Moutan Cortex charcoal by involving in the pathways related to complement, coagulation cascades and the platelet activation, particularly by activating FVIII, FX, FII and FXII and inhibiting SERPINC1. This study has demonstrated that Moutan Cortex charcoal may work as a hemostatic through the interaction between multiple-compounds and multiple-proteins, which provides the basis for further researches on the hemostasis mechanism of Moutan Cortex charcoal.


Assuntos
Carvão Vegetal/química , Carvão Vegetal/uso terapêutico , Medicamentos de Ervas Chinesas/química , Transtornos Hemorrágicos/tratamento farmacológico , Paeonia/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Proteínas/química
4.
J Pharm Biomed Anal ; 169: 151-158, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30861407

RESUMO

Moutan Cortex charcoal (MCC), the processed root bark of Paeonia suff ;ruticosa Andrews (Paeoniaceae), is a kind of traditional Chinese medicine (TCM) and has been used for treating blood-heat and hemorrhage(BHH)syndrome in China for thousands of years. In order to explore potential metabolic mechanism, 1H NMR-based metabonomics technique was applied to evaluate the effect of MCC on metabolic changes in plasma and urine of BHH rat models. Serum and urine samples were obtained from male SD rats with normal group, model group and MCC group for study. Based on 1H NMR spectra obtained from plasma and urine samples, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) models were capable of distinguishing the three group. And the 13 pharmacodynamic biomarkers of MCC were identified in the plasma and urine. The results showed that BHH induced great metabolic disorders in plasma and urine metabolisms. However, MCC could reverse the imbalanced metabolites by alanine, aspartate and glutamate metabolism and citrate cycle (TCA cycle) pathway, and its effect was also confirmed by the general signs and pharmacodynamics assessments. The results indicated that NMR-based metabolomic profiling method is sensitive and specific enough to evaluate the MCC efficacy and mechanism of action on BHH syndromes.


Assuntos
Carvão Vegetal/química , Carvão Vegetal/farmacologia , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Hemorragia/tratamento farmacológico , Paeonia/química , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Animais , Biomarcadores/sangue , Biomarcadores/urina , Análise Discriminante , Hemorragia/sangue , Hemorragia/urina , Temperatura Alta , Análise dos Mínimos Quadrados , Espectroscopia de Ressonância Magnética/métodos , Masculino , Medicina Tradicional Chinesa/métodos , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Plasma/química , Análise de Componente Principal/métodos , Ratos , Ratos Sprague-Dawley , Urina/química
5.
Int J Cancer ; 144(11): 2677-2682, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30255937

RESUMO

Kinase domain duplications of the epidermal growth factor receptor (EGFR-KDD) have been identified and implicated to be oncogenic in nonsmall cell lung cancers (NSCLCs). However, its prevalence and clinical contributions in lung cancer are largely unknown. Here, we conducted a multicenter record review of 10,759 NSCLC patients who underwent genetic testing using next-generation sequencing (NGS) targeting EGFR exons and the introns involved in EGFR-KDD rearrangements. EGFR-KDDs were identified in a total of 13 patients, which is approximately 0.12% of the total population reviewed, and also consisted of 0.24% (13/5394) of EGFR mutation-positive patients. A total of 85% of patients (11/13) were identified with the canonical EGFR-KDD duplication of exons 18-25, while the remaining two cases harbored duplications of EGFR exons 14-26 and exons 17-25, which have not been previously described. Importantly, none of the 13 patients had other coexisting driver mutations, highlighting the potential oncogenic role of this type of alteration. Three out of five patients who had exon 18-25 duplications showed partial antitumor responses to targeted therapies, while the other two patients demonstrated no clinical improvement. Furthermore, our data suggested that the EGFR T790 M mutation and EGFR amplification may represent the major resistance mechanisms against targeted therapies in tumors bearing EGFR-KDD. In summary, our findings provide valuable insight into the prevalence of EGFR-KDDs in NSCLCs and their clinical outcomes to targeted therapies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Domínios Proteicos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Análise Mutacional de DNA , Receptores ErbB/genética , Éxons/genética , Feminino , Amplificação de Genes , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos
6.
J Sep Sci ; 40(10): 2151-2160, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28371353

RESUMO

Raw Arecae Semen, the seed of Areca catechu L., as well as Arecae Semen Tostum and Arecae semen carbonisata are traditionally processed by stir-baking for subsequent use in a variety of clinical applications. These three Arecae semen types, important Chinese herbal drugs, have been used in China and other Asian countries for thousands of years. In this study, the sensory technologies of a colorimeter and sensitive validated high-performance liquid chromatography with diode array detection were employed to discriminate raw Arecae semen and its processed drugs. The color parameters of the samples were determined by a colorimeter instrument CR-410. Moreover, the fingerprints of the four alkaloids of arecaidine, guvacine, arecoline and guvacoline were surveyed by high-performance liquid chromatography. Subsequently, Student's t test, the analysis of variance, fingerprint similarity analysis, hierarchical cluster analysis, principal component analysis, factor analysis and Pearson's correlation test were performed for final data analysis. The results obtained demonstrated a significant color change characteristic for components in raw Arecae semen and its processed drugs. Crude and processed Arecae semen could be determined based on colorimetry and high-performance liquid chromatography with a diode array detector coupled with chemometrics methods for a comprehensive quality evaluation.


Assuntos
Areca/química , Medicamentos de Ervas Chinesas/análise , China , Cromatografia Líquida de Alta Pressão , Análise de Componente Principal , Controle de Qualidade , Sementes/química
7.
Pharmacogn Mag ; 12(45): 50-6, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27019561

RESUMO

BACKGROUND: Raw Moutan Cortex (RMC), derived from the root bark of Paeonia suffruticosa, and Processed Moutan Cortex (PMC) is obtained from RMC by undergoing a stir-frying process. Both of them are indicated for different pharmacodynamic action in traditional Chinese medicine, and they have been used in China and other Asian countries for thousands of years. OBJECTIVE: To establish a method to study the RMC and PMC, revealing their different chemical composition by fingerprint, qualitative, and quantitative ways. MATERIALS AND METHODS: High-performance liquid chromatography coupled with diode array detector and electrospray mass spectrometry (HPLC-DAD-ESIMS) were used for the analysis. Therefore, the analytes were separated on an Ultimate TM XB-C18 analytical column (250 mm × 4.6 mm, 5.0 µm) with a gradient elution program by a mobile phase consisting of acetonitrile and 0.1% (v/v) formic acid water solution. The flow rate, injection volume, detection wavelength, and column temperature were set at 1.0 mL/min, 10 µL, 254 nm, and 30°C, respectively. Besides, principal components analysis and the test of significance were applied in data analysis. RESULTS: The results clearly showed a significant difference among RMC and PMC, indicating the significant changes in their chemical compositions before and after the stir-frying process. CONCLUSION: The HPLC-DAD-ESIMS coupled with chemometrics analysis could be used for comprehensive quality evaluation of raw and processed Moutan Cortex. SUMMARY: The experiment study the RMC and PMC by HPLC-DAD-ESIMS couple with chemometrics analysis. The results of their fingerprints, qualitative, and quantitative all clearly showed significant changes in their chemical compositions before and after stir-frying processed. Abbreviation used: HPLC-DAD-ESIMS: High-performance Liquid Chromatography-Diode Array Detector-Electrospray Mass Spectrometry, RMC: Raw moutan cortex, PMC: Processed moutan cortex, TCM: Traditional Chinese medicine, PCA: Principal components analysis, LOD: Limit of detection, LOQ: Limit of quantitation, RSD: Relative standard deviation.

8.
J Sep Sci ; 38(17): 2945-52, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26174663

RESUMO

A sensitive, simple, and validated high-performance liquid chromatography with diode array detection and mass spectrometry detection method was developed for three ginger-based traditional Chinese herbal drugs, Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata. Chemometrics methods, such as principal component analysis, hierarchical cluster analysis, and analysis of variance, were also employed in the data analysis. The results clearly revealed significant differences among Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata, indicating variations in their chemical compositions during the processing, which may elucidate the relationship of the thermal treatment with the change of the constituents and interpret their different clinical uses. Furthermore, the sample consistency of Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata can also be visualized by high-performance liquid chromatography with diode array detection and mass spectrometry analysis followed by principal component analysis/hierarchical cluster analysis. The comprehensive strategy of liquid chromatography with mass spectrometry analysis coupled with chemometrics should be useful in quality assurance for ginger-based herbal drugs and other herbal medicines.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Análise de Alimentos/métodos , Gengibre/química , Espectrometria de Massas/métodos , Rizoma/química , China , Análise por Conglomerados , Medicamentos de Ervas Chinesas/química , Plantas Medicinais/química , Análise de Componente Principal , Controle de Qualidade
9.
Med Oncol ; 29(2): 644-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21516483

RESUMO

We report a rare case of a 38-year-old woman with metastatic colonic adenocarcinoma from primary lung adenocarcinoma detected by PET/CT. She underwent colonoscopy and adenocarcinoma was diagnosed in the pathology report, which was the same as that for lymph node biopsy from a left supraclavicular lymph node. We used immunohistochemistry to diagnose primary adenocarcinoma of the lung with colonic metastasis. Owing to mutation in exon 19 of EGFR gene, targeted therapy was given to her with a prescription of oral gefitinib for 1 month as first-line treatment. It was chosen to further treat the patient with chemotherapy and radiotherapy. As the patient was suffering from increasing coughing and sputum, radiotherapy and chemotherapy were subsequently cancelled. Since the general condition of the patient was relatively poor, Tarceva was therefore prescribed. The patient had lived for 5 more months since the diagnosis of metastatic colonic adenocarcinoma.


Assuntos
Adenocarcinoma/secundário , Neoplasias do Colo/patologia , Linfonodos/patologia , Adenocarcinoma/terapia , Adulto , Neoplasias do Colo/terapia , Colonoscopia , Feminino , Humanos , Imagem Multimodal , Tomografia por Emissão de Pósitrons , Prognóstico , Literatura de Revisão como Assunto , Tomografia Computadorizada por Raios X
10.
Thorac Cancer ; 2(4): 164-178, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27755845

RESUMO

BACKGROUND: Methylseleninic acid (MSA) has been implicated as a promising anticancer agent for lung cancer. However, the underlying molecular mechanism(s) responsible for MSA's action is not well understood. Our study aimed to examine the cellular effects of MSA on L9981 human high-metastatic large cell lung cancer cells and gain insights into its possible molecular mechanism(s) through a proteomic approach. METHODS: L9981 cells were exposed to MSA at different concentrations and time points. The effects of MSA on cell proliferation and apoptosis were detected by cell viability analyzer Vi-CELL and flow cytometric analysis, respectively. We analyzed the alterations in the proteome profile of L9981 cells induced by MSA using the 2-D difference in gel electrophoresis (2-D DIGE) and identified the differentially expressed proteins using a liquid chromatography system followed by tandem mass spectrometry (LC-MS/MS). RESULTS: We found that MSA inhibited cell proliferation in a dose-dependent manner and significantly induced early apoptosis in L9981 cells. 2-D DIGE showed that MSA induced significant changes (>1.29 fold) in the expression levels of 42 protein spots compared to the untreated control (P < 0.05). As identified by LC-MS/MS, proteins that underwent changes in response to MSA were related to various biological functions, including: (i) endoplasmic reticulum stress (upregulation of molecular chaperones like heat shock protein A5, protein disulfide-isomerase precursor, and calreticulin precursor); (ii) oxidative stress response/ thioredoxin system (decreased thioredoxin-like protein 1 and increased thioredoxin reductase 1); (iii) translation regulation (downregulation of translation factors like elongation factor 1-beta and eukaryotic translation initiation factor 6); (iv) mitochondrial bioenergetic function (upregulation of adenosine triphosphate synthase subunit beta and mitochondria); and (v) cell signal transduction regulation (decreased peptidyl-prolyl cis-trans isomerase A and 14-3-3 protein gamma). The protein and gene expression levels of those proteins of interest were further confirmed by Western blot and/or real-time reverse transcription polymerase chain reaction. CONCLUSION: Our results suggest that MSA may inhibit cell proliferation and induce apoptosis in lung cancer by modulating multiple targets involved in various crucial cellular processes.

11.
Med Sci Monit ; 16(8): CR357-64, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20671611

RESUMO

BACKGROUND: To analyze the prognostic significance of the genes casein kinase 2 alpha subunit (CSNK2A1), anti-apoptosis clone-11 (AAC-11), and tumor metastasis suppressor NME1 in completely resected non-small cell lung cancer (NSCLC) patients. MATERIAL/METHODS: Total RNA was extracted from 145 cases of completely resected, formalin-fixed, paraffin-embedded NSCLC tissues. mRNA expression levels of CSNK2A1, AAC-11, and NME1 were detected by real-time reverse-transcriptase polymerase chain reaction. Univariate and multivariate survival analyses were used to identify factors related to prognosis. RESULTS: A correlation between CSNK2A1 and AAC-11 mRNA expression levels (rs=0.366, p=0.000) was found. Univariate analysis showed that high expression of CSNK2A1 and AAC-11 was predictive of poor prognosis in NSCLC patients (p=0.029 and 0.044, respectively), especially when expression levels of both genes were concomitantly high (p=0.007). Multivariate Cox regression analysis showed that high expression of CSNK2A1, or concomitantly high expression of CSNK2A1 and AAC-11, are independent prognostic factors of poor survival in NSCLC patients. However, NME1 mRNA expression level did not significantly influence survival in NSCLC patients. CONCLUSIONS: This retrospective study indicates that CSNK2A1 and AAC-11, especially in combination, are useful prognosis markers in NSCLC patients after complete resection, independent of lymph node metastasis status.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Caseína Quinase II/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Nucleosídeo NM23 Difosfato Quinases/genética , Proteínas Nucleares/genética , Adulto , Idoso , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Caseína Quinase II/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas Nucleares/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Surg Today ; 39(4): 332-4, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19319642

RESUMO

Mediastinal solitary fibrous tumors (SFTs) are rarely found in adults and there are few reports describing primary mediastinal SFT invading the diaphragm. We report the case of a 47-year-old woman with a large right inferior mediastinal SFT. Magnetic resonance imaging showed the tumor invading the right lower lobe of the lung and the right hemidiaphragm, with displacement of the inferior vena cava (IVC) and right lobe of the liver. Angiogram showed IVC stenosis. To our knowledge, this is the first report of complete resection of the tumor combined with right lower lobectomy of the lung and partial resection and reconstruction of the right diaphragm with a Dacron flap.


Assuntos
Diafragma/patologia , Neoplasias do Mediastino/patologia , Neoplasias do Mediastino/cirurgia , Tumores Fibrosos Solitários/patologia , Tumores Fibrosos Solitários/cirurgia , Angiografia , Feminino , Humanos , Imagem por Ressonância Magnética , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Polietilenotereftalatos , Toracotomia , Tomografia Computadorizada por Raios X
14.
Biochem Biophys Res Commun ; 371(3): 425-30, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18440302

RESUMO

Lung cancer is the leading cause of cancer death in both men and women. Tumor metastasis is an essential aspect of lung cancer progression. nm23-H1 is a metastasis suppressor gene. The molecular mechanism by which nm23-H1 suppresses the metastasis is still unclear. Here, we compared the gene expression profile of human large cell lung cancer cell line NL9980 by nm23-H1 gene silencing with that of negative control cells to comprehensively investigate nm23-H1-mediated changes in gene expression of NL9980 cells. Microarray assay revealed that expression of 733-known genes (1.9%, 733/38,500) were altered in response to nm23-H1 gene silencing, including 466 upregulated genes and 267 downregulated. real-time PCR assay of the expression changes indicated that 81.82% (45/55) of verified genes were consistent with that observed in microarray assay. The upregulated genes included MMP-1, -2, SNAI2, CXCL1, 2, 3, PAI-2, while the downregulated genes included cystatin B, TIMP-2, E-cadherin, centrin-2, all of which have been associated with tumor metastasis. Furthermore, we confirmed by Western blot that the expression of MMP-1 and -2 were significantly increased while that of cystatin B was dramatically decreased in NL9980-nm23-H1 silencing cells. The NL9980-nm23-H1 silencing cells exhibited significantly more S phase growth and invasive ability. Thus, silencing of nm23-H1 gene caused metastasis-related gene expression changes in lung cancer cells. The knockdown of nm23-H1 expression may change the lung cancer cells to a more invasive phenotype through alteration in the expression of a set of genes.


Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Linhagem Celular Tumoral , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno/genética
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