RESUMO
HOTAIR is a long non-coding RNA that interacts with the polycomb repressive complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition process, and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in human cancer cells. miR-141 binds to HOTAIR in a sequence-specific manner and suppresses HOTAIR expression and functions, including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Ago2 (Argonaute2) complex, and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner.
Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Apoptose/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Células HT29 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , MicroRNAs/metabolismo , Mutação , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , RNA Longo não Codificante/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Lung cancer is clearly the primary cause of cancer-related deaths worldwide. Recent molecular-targeted strategy has contributed to improvement of the curative effect of adenocarcinoma of the lung. However, such current treatment has not been developed for squamous cell carcinoma (SCC) of the disease. The new genome-wide RNA analysis of lung-SCC may provide new avenues for research and the development of the disease. Our recent microRNA (miRNA) expression signatures of lung-SCC revealed that clustered miRNAs miR-1/133a were significantly reduced in cancer tissues. Here, we found that restoration of both mature miR-1 and miR-133a significantly inhibited cancer cell proliferation, migration and invasion. Coronin-1C (CORO1C) was a common target gene of the miR-1/133a cluster, as shown by the genome-wide gene expression analysis and the luciferase reporter assay. Silencing of CORO1C gene expression inhibited cancer cell proliferation, migration and invasion. Furthermore, CORO1C-regulated molecular pathways were categorized by using si-CORO1C transfectants. Further analysis of novel cancer signaling pathways modulated by the tumor-suppressive cluster miR-1/133a will provide insights into the molecular mechanisms of lung-SCC oncogenesis and metastasis.
Assuntos
Carcinoma de Células Escamosas/genética , Movimento Celular/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Idoso , Idoso de 80 Anos ou mais , Azacitidina/farmacologia , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Família Multigênica , Invasividade Neoplásica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Our recent studies of the microRNA (miRNA) expression signature in prostate cancer (PCa) indicated that miRNA-218 (miR-218) was significantly downregulated in clinical specimens, suggesting that miR-218 might act as a tumor-suppressive miRNA in PCa. The aim of the present study was to investigate the functional significance of miR-218 in PCa and to identify novel miR-218-regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis. Restoration of miR-218 in PCa cell lines (PC3 and DU145) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that LIM and SH3 protein 1 (LASP1) is a potential target of miR-218 regulation. LASP1 is a cytoskeletal scaffold protein that plays critical roles in cytoskeletal organization and cell migration. Luciferase reporter assays showed that miR-218 directly regulated expression of LASP1. Moreover, downregulating the LASP1 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of LASP1 was upregulated in cancer tissues. We conclude that loss of tumor-suppressive miR-218 enhanced cancer cell migration and invasion in PCa through direct regulation of LASP1. Our data on pathways regulated by tumor-suppressive miR-218 provide new insight into the potential mechanisms of PCa oncogenesis and metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Movimento Celular/genética , Proliferação de Células , Proteínas do Citoesqueleto/genética , Humanos , Proteínas com Domínio LIM/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Our recent study of microRNA (miRNA) expression signature of prostate cancer (PCa) has revealed that the microRNA-143/145 (miR-143/145) cluster is significantly downregulated in cancer tissues, suggesting that these cluster miRNAs are candidate tumor suppressors. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in PCa cells and to identify novel targets regulated by these cluster miRNAs in PCa. Restoration of miR-143 or miR-145 in PCa cell lines (PC3 and DU145) revealed that these miRNAs significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that Golgi membrane protein 1 (GOLM1) resembling a type II golgi transmembrane protein was a potential target of miR-143/145 cluster target gene. Gene expression studies and luciferase reporter assays showed that GOLM1 was directly regulated by the miR-143/145 cluster. Silencing of GOLM1 resulted in significant inhibition of cell migration and invasion in PCa cells. Furthermore, the expression of GOLM1 was upregulated in cancer tissues by immunohistochemistry. Loss of the tumor-suppressive miR-143/145 cluster enhanced cancer cell migration and invasion in PCa through directly regulating GOLM1. Our data on target genes regulated by the tumor-suppressive miR-143/145 cluster provide new insights into the potential mechanisms of PCa oncogenesis and metastasis.
Assuntos
Movimento Celular , Regulação da Expressão Gênica , Proteínas de Membrana/biossíntese , MicroRNAs/metabolismo , Família Multigênica , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , RNA Neoplásico/metabolismo , Idoso , Linhagem Celular Tumoral , Humanos , Masculino , Proteínas de Membrana/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Neoplásico/genéticaRESUMO
The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C â G, Arg â Gly, rs10012), 119 (G â T, Ala â Ser, rs1056827), 432 (C â G, Leu â Val, rs1056836), 449 (C â T, Asp, rs1056837), and 453 (A â G, Asn â Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/genética , Citocromo P-450 CYP1B1/genética , Predisposição Genética para Doença/genética , Neoplasias Renais/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/enzimologia , Genótipo , Humanos , Neoplasias Renais/enzimologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Fatores de RiscoRESUMO
Our recent studies of microRNA (miRNA) expression signatures have indicated that the miR-143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in cancer cells and to identify novel molecular targets of the miR-143/145 cluster in renal cell carcinoma (RCC). The expression levels of miR-143 and miR-145 were significantly downregulated in RCC tissues compared with adjacent non-cancerous tissues. A significant positive correlation was recognized between miR-143 and miR-145 expression. Restoration of mature miR-143 or miR-145 in 786-O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR-143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis showed that the hexokinase-2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells, was a candidate target of the miR-143/145 cluster. Luciferase reporter assays showed that both miR-143 and miR-145 directly regulated HK2. In RCC clinical specimens, the expression of HK2 was significantly higher in cancer tissues than in non-cancerous tissues. Silencing HK2 suppressed RCC cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. Thus, our data showed that loss of the tumor-suppressive miR-143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2.
Assuntos
Carcinoma de Células Renais/genética , Hexoquinase/metabolismo , Neoplasias Renais/genética , MicroRNAs/metabolismo , Sítios de Ligação/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Genes Supressores de Tumor , Hexoquinase/genética , Humanos , Neoplasias Renais/patologia , Luciferases , MicroRNAs/genética , Invasividade Neoplásica , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/enzimologia , Catecol O-Metiltransferase/metabolismo , Estradiol/análogos & derivados , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Catecol O-Metiltransferase/biossíntese , Catecol O-Metiltransferase/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios de Catecol , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Regulação para Cima/efeitos dos fármacosRESUMO
We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate many oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3'-untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3'UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , MicroRNAs/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Caspase 3/biossíntese , Caspase 7/biossíntese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Proteínas Nucleares/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Processamento de Serina-ArgininaRESUMO
microRNAs (miRNAs) have key roles in human tumorigenesis, tumor progression and metastasis. miRNAs are aberrantly expressed in many human cancers and can function as tumor suppressors or oncogenes that target many cancer-related genes. This study seeks to identify novel miRNA-regulated molecular pathways in prostate cancer (PCa). The miRNA expression signature in clinical specimens of PCa showed that 56 miRNAs were significantly downregulated in PCa compared with non-PCa tissues. We focused on the top four downregulated miRNAs (miR-187, miR-205, miR-222 and miR-31) to investigate their functional significance in PCa cells. Expression levels of these four miRNAs were validated in PCa specimens (15 PCa tissues and 17 non-PCa tissues) to confirm that they were significantly reduced in these PCa tissues. Gain-of-function analysis demonstrated that miR-222 and miR-31 inhibited cell proliferation, invasion and migration in PCa cell lines (PC3 and DU145), suggesting that miR-222 and miR-31 may act as tumor suppressors in PCa. Genome-wide gene expression analysis using miR-222 or miR-31 transfectants to identify the pathways they affect showed that many cancer-related genes are regulated by these miRNAs in PC3 cells. Identification and categorization of the molecular pathways regulated by tumor suppressive miRNAs could provide new information about the molecular mechanisms of PCa tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias da Próstata/genética , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Simulação por Computador , Genes Supressores de Tumor , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologia , Reprodutibilidade dos TestesRESUMO
A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.
Assuntos
Biomarcadores Tumorais/urina , MicroRNAs/urina , Neoplasias da Bexiga Urinária/patologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Curva ROC , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urinaRESUMO
BACKGROUND: MiR-145 is down-regulated in various human cancers. We previously demonstrated that some actin-binding proteins were targeted by several microRNAs (miRNAs), including miR-145, in bladder and prostate cancer (CaP). The aim of this study is to determine a novel oncogenic gene targeted by miR-145 by focusing on actin-binding proteins in CaP. METHODS: We focused on the SWAP switching B-cell complex 70 kDa subunit (SWAP70), which is an F-actin binding protein involved in activating B-cell transformation. A luciferase reporter assay was used to identify the actual binding sites between miR-145 and SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-SWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS: Molecular target searches of this miRNA and the luciferase reporter assay showed that SWAP70 was directly regulated by miR-145. Silencing of SWAP70 studies demonstrated significant inhibitions of cell migration and invasion in CaP cell lines. The SWAP70 positive-staining was significantly higher in percentage in the CaP than in benign prostate hyperplasia tissue. CONCLUSIONS: Down-regulation of miR-145 was a frequent event in CaP, and it may have a tumor suppressive function. SWAP70 may be a target of miR-145, and it might have a potential oncogenic function. The novel molecular networks though which miR-145 acts, may provide new insights into the underlying molecular mechanisms of CaP.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Luciferases/genética , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Antígenos de Histocompatibilidade Menor , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.
Assuntos
Carcinoma de Células Escamosas/genética , Moléculas de Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Western Blotting , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proto-Oncogenes/genética , Interferência de RNA , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Células Escamosas de Cabeça e Pescoço , TransfecçãoRESUMO
MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in esophageal squamous cell carcinoma (ESCC) was performed using the miRNA expression signatures obtained from ESCC clinical specimens. A subset of 15 miRNAs was significantly downregulated in ESCC. A comparison of miRNA signatures from ESCC and our previous report identified 4 miRNAs that are downregulated in common (miR-145, miR-30a-3p, miR-133a and miR-133b), suggesting that these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion in ESCC cells. These miRNAs (miR-145, miR-133a and miR-133b), which have conserved sequences in the 3'UTR of FSCN1 (actin-binding protein, Fascin homolog 1), inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.
Assuntos
Carcinoma de Células Escamosas/genética , Proteínas de Transporte/genética , Neoplasias Esofágicas/genética , Genes Supressores de Tumor/fisiologia , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Regiões 3' não Traduzidas/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Adesão Celular , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7.
Assuntos
MicroRNAs/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Algoritmos , Sequência de Bases , Divisão Celular , Feminino , Humanos , Masculino , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Bexiga Urinária/patologiaRESUMO
In our microarray screening of methylated genes in bladder cancer (BC), the collagen type 1 alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.
Assuntos
Movimento Celular , Proliferação de Células , Colágeno/genética , Ilhas de CpG/genética , Metilação de DNA , Neoplasias da Próstata/patologia , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Adesão Celular , Colágeno/metabolismo , Colágeno Tipo I , Decitabina , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genéticaRESUMO
PURPOSE: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels. MATERIALS AND METHODS: We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth. RESULTS: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma. CONCLUSIONS: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/genética , Ilhas de CpG/genética , Metilação de DNA , Neoplasias Renais/genética , Regiões Promotoras Genéticas/genética , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Humanos , PrognósticoRESUMO
A disintegrin and metalloproteases (ADAM) are cell membrane-anchored proteins with potential implications for the metastasis of human cancer cells via cell adhesion and protease activities. In prostate cancer (PC), the ADAM-10 protein showed a nuclear localization whereas in benign prostate hypertrophy (BPH) it was predominantly bound to the cell membrane. We hypothesized that the pathogenesis and progression of PC are attributable to the nuclear translocation of ADAM-10. Immunoblotting revealed that after 5alpha-dihydrotestosterone treatment, a 60-kDa active form of ADAM-10 was increased in the nuclear fraction but decreased in the cell membrane and cytoplasmic fractions of human androgen-dependent PC cells. Immunocytochemistry revealed that after 5alpha-dihydrotestosterone treatment, the ADAM-10 protein was translocated from the cell membrane to the nucleus. Coimmunoprecipitation of androgen receptor and ADAM-10 was detected in the nuclear fraction but not in the cell membrane and cytoplasmic fractions. Immunohistochemical study of 64 PC and 20 BPH samples showed that the intensity of ADAM-10 staining was significantly higher in the nuclei of PC cells than in the nuclei of BPH cells (P < 0.0001). It was also significantly lower in the cell membrane of PC cells than in the cell membrane of BPH cells (P = 0.0017). Nuclear staining intensity was significantly correlated with the clinical T-factor (P = 0.004), the Gleason score (P < 0.0001) and preoperative prostate-specific antigen levels (P = 0.0061). ADAM-10 small interfering RNA transfectants showed a significant decrease in cell growth compared to the controls. Our results suggest that in human PC, the nuclear translocation of ADAM-10 coupled with the androgen receptor is involved in tumor growth and progression.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/patologia , Proteínas ADAM/genética , Proteína ADAM10 , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/genética , Biópsia , Linhagem Celular Tumoral , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Primers do DNA , Di-Hidrotestosterona/farmacologia , Progressão da Doença , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 µM) significantly suppressed proliferation, migration, invasion and induced apoptosis of T24 and UM-UC-3 human bladder cancer cells. Silibinin down-regulated the actin cytoskeleton and phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathways in these cancer cell lines. These pathways were found to crosstalk through RAS cascades. We found that silibinin suppressed levels of trimethylated histone H3 lysine 4 and acetylated H3 at the KRAS promoter. Furthermore, silibinin targets long non-coding RNA: HOTAIR and ZFAS1, which are known to play roles as oncogenic factors in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of actin cytoskeleton and PI3K/Akt pathways and thus suppresses bladder cancer growth and progression.
RESUMO
In developed countries, endometrial cancer (EC) is the most common malignancy among women. Unopposed estrogen therapy, obesity, nulliparity, diabetes mellitus and arterial hypertension have been linked to an increased risk of EC. However, the molecular mechanisms of EC oncogenesis and metastasis have not yet been fully elucidated. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-1/133a (miR1/133a) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR1/133a cluster in EC cells is still unknown. Thus, the aim of this study was to investigate the functional significance of the miR1/133a cluster and its regulated molecular targets, with an emphasis on the contributions of miR1/133a to EC oncogenesis and metastasis. We found that the expression levels of miR1 and miR133a were significantly reduced in EC tissues. Moreover, restoration of mature miR1 or miR133a miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs act as tumor suppressors. Prediction of miRNA targets revealed that phosphodiesterase 7A (PDE7A) was a potential target gene regulated by both miR1 and miR133a. PDE7A was confirmed to be overexpressed in EC clinical specimens and silencing of PDE7A significantly inhibited cancer cell migration and invasion. Our data demonstrated that downregulation of the miR1/133a cluster promoted cancer cell migration and invasion via overexpression of PDE7A in EC cells. Elucidation of the molecular networks regulated by tumor-suppressive miRNAs will provide insights into the molecular mechanisms of EC oncogenesis and metastasis.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Família Multigênica , Invasividade NeoplásicaRESUMO
Here, we found that members of the microRNA-29 family (miR-29a/b/c; "miR-29s") were significantly reduced in clear cell renal cell carcinoma (ccRCC) tissues, suggesting that they functioned as tumour suppressors. Restoration of all mature members of the miR-29 family inhibited cancer cell proliferation, migration and invasion. LOXL2 was a direct target gene of miR-29s, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed LOXL2 was confirmed in ccRCC clinical specimens, and silencing of LOXL2 inhibited cancer cell migration and invasion in ccRCC cell lines. Our data demonstrated that the miR-29s-LOXL2 axis contributed to cancer cell migration and invasion in ccRCC.