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1.
Lab Invest ; 92(7): 1013-9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22488152

RESUMO

We previously reported frequent truncating mutations of the RNA-binding protein gene, La ribonucleoprotein domain family, member-7 (LARP7) in gastric cancers (GCs) with frequent microsatellite instability. LARP7 negatively regulates positive transcription elongation factor-b (p-TEFb) by binding to and stabilizing 7sk RNA. p-TEFb has been linked to proliferation and de-differentiation in various tissues. Therefore, we reasoned that loss of LARP7 may contribute to gastric tumorigenesis. In this study, we evaluated LARP7 mRNA expression in 18 GCs, their corresponding non-neoplastic gastric tissues (N(GC)), and 18 normal gastric tissues from healthy individuals (N(N)). We also assessed the effects of transient small interfering (siRNA)-mediated LARP7 knockdown in immortalized non-neoplastic gastric epithelial cells. LARP7 mRNA was significantly decreased in GCs (median 2.5) relative to N(N)s (median 14.9, P<0.01) as well as relative to their corresponding N(GC)s (median 8.1, P<0.01). Transfection of an siRNA directed against LARP7 (anti-LARP7 siRNA) into non-neoplastic gastric epithelial cells decreased 7sk levels by 72% relative to a control siRNA (P<0.01). Furthermore, anti-LARP7 siRNA transfection increased cell proliferation by 23% (P<0.01) and cell migration by 22% (P<0.001) relative to control siRNA transfection. Taken together, these findings suggest that LARP7 downregulation occurs early during gastric tumorigenesis and may promote gastric tumorigenesis via p-TEFb dysregulation.


Assuntos
Genes Supressores de Tumor , Ribonucleoproteínas/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
2.
Hepatology ; 54(6): 2089-98, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21809359

RESUMO

UNLABELLED: MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494. Up-regulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 down-regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3'-untranslated region of cyclin-dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in vivo. CONCLUSION: Our findings demonstrate that miR-494 is down-regulated in CCA and that its up-regulation induces cancer cell growth retardation through multiple targets involved in the G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics.


Assuntos
Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Colangiocarcinoma/genética , MicroRNAs/genética , Animais , Neoplasias dos Ductos Biliares/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/biossíntese , Transplante de Neoplasias , Transfecção , Transplante Heterólogo
3.
Int J Cancer ; 129(9): 2134-46, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21170987

RESUMO

Polo-like kinase 1 (PLK1) is overexpressed in various human cancers. However, the biological functions and the post-transcriptional regulations of PLK1 in esophageal cancer (EC) are still unknown. The purposes of our study are to determine whether PLK1 can be a molecular target of EC therapy and to identify a microRNA (miRNA) targeting PLK1. We performed loss-of-function and gain-of-function experiments regarding cell proliferation, cell cycle, apoptosis, in vivo tumor formation and luciferase reporter assays, using siRNAs against PLK1 and miRNA. PLK1 protein was expressed in all 11 EC cell lines, but not in normal esophageal epithelial cells (HEEpiC). Knockdown of PLK1 in EC cells induced G2/M arrest (p < 0.001) in cell cycle assay and reduced cell proliferation (p = 0.019) and tumor formation ability in vivo (p < 0.0001). MiR-593*, identified as a miRNA targeting PLK1 by a database search, was less expressed especially in six EC cell lines than HEEpiC cells. Moreover, miR-593* expression level was inversely correlated with PLK1 mRNA level in 48 clinical tissue specimens of EC (p = 0.006). Introduction of synthetic miR-593* suppressed PLK1 expression by 69-73%, reduced cell proliferation (p = 0.008) and increased cell proportion of G2/M phase (p = 0.01) in HSA/c (an EC cells), whereas a miR-593* inhibitor upregulated PLK1 expression by 11-55%. Additionally, luciferase assay demonstrated that miR-593* interacted two binding sites in the PLK1 3'-UTR and reduced 56.8-71.5% of luciferase activity by degrading luciferase mRNA in HSA/c cells. In conclusion, PLK1 is post-transcriptionally regulated by miR-593* and could be a promising molecular target for EC treatment.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Estabilidade de RNA , RNA Mensageiro/metabolismo , Quinase 1 Polo-Like
4.
Gastroenterology ; 136(5): 1689-700, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19422085

RESUMO

BACKGROUND & AIMS: Barrett's esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known. METHODS: Esophageal cultured cells (HEEpiC, QhTRT, ChTRT, GihTRT, and OE-33) and esophageal tissues (22 normal epithelia, 24 BE, and 22 EAC) were studied. MiR microarrays and quantitative reverse-transcription polymerase chain reaction (RT-PCR) were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study copy number variation at the miR-106b-25 polycistron and MCM7 gene locus on chromosome 7q22.1. In vitro cell proliferation, cell cycle, and apoptosis assays and in vivo tumorigenesis experiments were performed to elucidate biologic effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct messenger RNA (mRNA) targeting by the miR-106b-25 polycistron. RESULTS: The miR-106b-25 polycistron exerted potential proliferative, antiapoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs-93 and -106b targeted and inhibited p21, whereas miR-25 targeted and inhibited Bim. This polycistron was upregulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7. In addition, miRs-93 and -106b decreased p21 mRNA, whereas miR-25 did not alter Bim mRNA, suggesting the following discrete miR effector mechanisms: (1) for p21, mRNA degradation; (2) for Bim, translational inhibition. CONCLUSIONS: The miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression and proliferation via suppression of 2 target genes: p21 and Bim.


Assuntos
Adenocarcinoma/genética , Proteínas Reguladoras de Apoptose/genética , Esôfago de Barrett/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Esofágicas/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , MicroRNAs/genética , Oncogenes/fisiologia , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional , Adenocarcinoma/metabolismo , Animais , Apoptose , Esôfago de Barrett/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Células Cultivadas , Neoplasias Esofágicas/metabolismo , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
5.
Hepatology ; 49(5): 1595-601, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19296468

RESUMO

UNLABELLED: Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR-21 was overexpressed in CCAs. miR-21 differential expression in these 10 specimens was verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the receiver operating characteristic curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA levels of TIMP3 were significantly lower in CCAs than in normals. CONCLUSIONS: MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Colangiocarcinoma/metabolismo , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J Clin Med ; 4(9): 1713-28, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26343737

RESUMO

Profound changes in microRNA (miR) expression levels are frequently found in liver cancers compared to the normal liver. In this study, we evaluate the expression of miR-224 in human HCC and CCA, as well as its downstream targets and affected pathways. We show that miR-224 is upregulated in a large cohort of human CCA, similar to its upregulation in human HCC. For the purpose of studying the roles of miR-224 in HCC and CCA, we enforced miR-224 expression in cells. mRNA arrays followed by Ingenuity Pathway Analysis (IPA)-identified putative molecules and pathways downstream of miR-224. Phenotypically, we report that enforced expression of miR-224 increases the growth rate of normal cholangiocytes, CCA cell lines, and HCC cell lines. In addition, we identified, in an unbiased fashion, that one of the major biologic processes affected by miR-224 is Gap1 (G1) to Synthesis (S) transition checkpoint release. We next identified p21, p15, and CCNE1 as downstream targets of miR-224 and confirmed the coordinated downregulation results in the increased phosphorylation of Retinoblastoma (Rb) with resulting G1/S checkpoint release. Our data suggest that miR-224 is a master regulator of cell cycle progression, and that its overexpression results in G1/S checkpoint release followed by accelerated cell growth.

8.
Oncotarget ; 6(8): 5666-77, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25686840

RESUMO

The complex regulation of tumor suppressive gene and its pseudogenes play key roles in the pathogenesis of hepatocellular cancer (HCC). However, the roles played by pseudogenes in the pathogenesis of HCC are still incompletely elucidated. This study identifies the putative tumor suppressor INTS6 and its pseudogene INTS6P1 in HCC through the whole genome microarray expression. Furthermore, the functional studies - include growth curves, cell death, migration assays and in vivo studies - verify the tumor suppressive roles of INTS6 and INTS6P1 in HCC. Finally, the mechanistic experiments indicate that INTS6 and INTS6P1 are reciprocally regulated through competition for oncomiR-17-5p. Taken together, these findings demonstrate INTS6P1 and INTS6 exert the tumor suppressive roles through competing for oncomiR-17-5p. Our investigation of this regulatory circuit reveals novel insights into the underlying mechanisms of hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Pseudogenes , Proteínas Ribossômicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Ligação Competitiva , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Genes Supressores de Tumor , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA , Proteínas Ribossômicas/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo
9.
Inflamm Bowel Dis ; 19(3): 471-80, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23399735

RESUMO

BACKGROUND: The development of colon cancer represents a major complication in patients with inflammatory bowel disease (IBD). The importance of microRNAs (miRs) in carcinogenesis is becoming clearer because miRs have been implicated in the regulation of cancer-related cellular processes to include apoptosis, differentiation, cell cycle progression, and immune function. In the current study, we sought to identify miR dysregulation specific to progression along the normal-inflammation-cancer axis in colonic specimens from patients with IBD. METHODS: MiR microarrays and quantitative reverse transcription PCR were used to detect and confirm dysregulated miRs. Receiver operating characteristic curve analysis was applied to evaluate the potential use of miR-224 as a neoplastic disease marker in IBD. For miR-224 target messenger RNA (mRNA) identification, mRNA microarrays were employed in combination with bioinformatic analyses, Western blotting, and luciferase activity measurements. RESULTS: We identified 30 miRs that were differentially expressed between chronically inflamed mucosae and cancers arising from IBD tissues. MiR-224 levels increased successively at each stage of IBD progression and accurately discriminated cancers from normal or chronically inflamed IBD tissues. Moreover, mRNA arrays combined with bioinformatic analyses suggested the participation of miR-224 in cell cycle regulation. Subsequently, cell cycle experiments indicated that miR-224 regulates the G1-S checkpoint. Finally, in silico prediction analyses, confirmed by Western blotting and luciferase assays, identified p21 as a specific direct mRNA target of miR-224. CONCLUSIONS: These findings reveal miR dysregulation specific to IBD-associated colorectal carcinoma. MiR-224 is overexpressed in IBD cancers and targets p21, a key cell cycle regulator. Moreover, these results establish the participation of miR-224 in IBD carcinogenesis.


Assuntos
Neoplasias do Colo/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Doenças Inflamatórias Intestinais/complicações , MicroRNAs/metabolismo , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Estudos de Coortes , Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Progressão da Doença , Citometria de Fluxo , Marcadores Genéticos , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Dig Liver Dis ; 44(7): 589-96, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22464652

RESUMO

BACKGROUND: A thorough understanding of gastric cancer at the molecular level is urgently needed. One prominent oncogenic microRNA, miR-21, was previously reported to be upregulated in gastric cancer. METHODS: We performed an unbiased search for downstream messenger RNA targets of miR-21, based on miR-21 dysregulation, by using human tissue specimens and the MKN28 human gastric carcinoma cell line. Molecular techniques include microRNA microarrays, cDNA microarrays, qRT-PCR for miR and mRNA expression, transfection of MKN28 with miR-21 inhibitor or Serpini1 followed by Western blotting, cell cycle analysis by flow cytometry and luciferase reporter assay. RESULTS: This search identified Serpini1 as a putative miR-21 target. Luciferase assays demonstrated direct interaction between miR-21 and Serpini1 3'UTR. miR-21 and Serpini1 expression levels were inversely correlated in a subgroup of gastric cancers, suggesting a regulatory mechanism that included both of these molecules. Furthermore, Serpini1 induced growth retardation of MKN28 and induced vigorous G1/S arrest suggesting its potential tumour-suppressive function in the stomach. CONCLUSION: Taken together, these data suggest that in a subgroup of gastric cancers, miR-21 is upregulated, inducing downregulation of Serpini1, which in turn releases the G1-S transition checkpoint, with the end result being increased tumour growth.


Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neuropeptídeos/genética , RNA Mensageiro/genética , Serpinas/genética , Neoplasias Gástricas/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , MicroRNAs/metabolismo , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Serpinas/metabolismo , Neoplasias Gástricas/metabolismo , Transfecção , Regulação para Cima , Neuroserpina
11.
Inflamm Bowel Dis ; 18(4): 641-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21830278

RESUMO

BACKGROUND: CpG island (CGI) hypermethylation at discrete loci is a prevalent cancer-promoting abnormality in sporadic colorectal carcinomas (S-CRCs). We investigated genome-wide CGI methylation in inflammatory bowel disease (IBD)-associated CRCs (IBD-CRCs). METHODS: Methylation microarray analyses were conducted on seven IBD-CRCs, 17 S-CRCs, and eight normal control colonic tissues from patients without CRC or IBD. CGI methylator phenotype (CIMP), a surrogate marker for widespread cancer-specific CGI hypermethylation, was examined in 30 IBD-CRCs and 43 S-CRCs. RESULTS: The genome-wide CGI methylation pattern of IBD-CRCs was CIMP status-dependent. Based on methylation array data profiling of all autosomal loci, CIMP(+) IBD-CRCs grouped together with S-CRCs, while CIMP(-) IBD-CRCs grouped together with control tissues. CIMP(-) IBD-CRCs demonstrated less methylation than did age-matched CIMP(-) S-CRCs at autosomal CGIs (z-score -0.17 vs. 0.09, P = 3 × 10(-3)) and CRC-associated hypermethylation target CGIs (z-score -0.43 vs. 0.68, P = 1 × 10(-4)). Age-associated hypermethylation target CGIs were significantly overrepresented in CGIs that were hypermethylated in S-CRCs (P = 1 × 10(-192)), but not in CGIs that were hypermethylated in IBD-CRCs (P = 0.11). In contrast, KRAS mutation prevalence was similar between IBD-CRCs and S-CRCs. Notably, CIMP(+) prevalence was significantly higher in older than in younger IBD-CRC cases (50.0 vs. 4.2, P = 0.02), but not in S-CRC cases (9.7 vs. 16.7, P = 0.92). CONCLUSIONS: Cancer-specific CGI hypermethylation and age-associated CGI hypermethylation are diminished in IBD-CRCs relative to S-CRCs, while the KRAS mutation rate is comparable between these cancers. CGI hypermethylation appears to play only a minor role in IBD-associated carcinogenesis. We speculate that aging, rather than inflammation per se, promotes CIMP(+) CRCs in IBD patients.


Assuntos
Carcinoma/genética , Neoplasias Colorretais/genética , Ilhas de CpG , Metilação de DNA , Doenças Inflamatórias Intestinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/etiologia , Colo/metabolismo , Neoplasias Colorretais/etiologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/complicações , Masculino , Pessoa de Meia-Idade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/genética
12.
Inflamm Bowel Dis ; 17(1): 221-31, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20848542

RESUMO

BACKGROUND: Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) expression has been linked to carcinogenesis; however, no reports document a relationship between IBD-related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal-inflammation-cancer axis. METHODS: miR microarrays and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR-31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification. RESULTS: Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR-31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR-31. CONCLUSIONS: Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR-31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that coexist in IBD patients. The novel effect of miR-31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Colo/patologia , Neoplasias Colorretais/etiologia , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/genética , MicroRNAs/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Luciferases/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Endocr Relat Cancer ; 18(4): 465-78, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21636702

RESUMO

DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon. We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to eight non-neoplastic colonic mucosae (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, nine adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs). A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (P<0.05 by receiver operator characteristic curve analysis); methylation of visual system homeobox 2 (VSX2) achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, P<1×10(-6)), followed by BEN domain containing 4 (BEND4), neuronal pentraxin I (NPTX1), ALX homeobox 3 (ALX3), miR-34b, glucagon-like peptide 1 receptor (GLP1R), BTG4, homer homolog 2 (HOMER2), zinc finger protein 583 (ZNF583), and gap junction protein, gamma 1 (GJC1). Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (P<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (P<1×10(-4)). In conclusion, systematic methylome-wide analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.


Assuntos
Adenoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Metilação de DNA , Adenoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Colo/metabolismo , Neoplasias Colorretais/diagnóstico , DNA de Neoplasias/genética , Epigenômica , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Reto/metabolismo
14.
PLoS One ; 4(11): e8002, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19956721

RESUMO

BACKGROUND: Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11 (CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region. METHODOLOGY/PRINCIPAL FINDINGS: Levels of CLDN11 methylation and mRNA expression were measured in primary gastric cancer tissues, noncancerous gastric mucosae, and cell lines of gastric origin using quantitative methylation-specific PCR (qMSP) and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2'-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness. CONCLUSIONS/SIGNIFICANCE: These data suggest that hypermethylation of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteínas do Tecido Nervoso/genética , Neoplasias Gástricas/metabolismo , Azacitidina/análogos & derivados , Azacitidina/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular , Claudinas , Metilação de DNA , Decitabina , Epigênese Genética , Humanos , Invasividade Neoplásica , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Cancer Res ; 69(10): 4112-5, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19435894

RESUMO

Esophageal adenocarcinoma risk in Barrett's esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of eight BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors and 50 progressors using real-time quantitative methylation-specific PCR. Progressors were significantly older than nonprogressors (70.6 versus 62.5 years; P < 0.001). We evaluated a linear combination of the eight markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUC) were high in the 2-year, 4-year, and combined data models (0.843, 0.829, and 0.840; P < 0.001, <0.001, and <0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age versus age alone were substantial (Delta-AUC = 0.152, 0.114, and 0.118, respectively) in all 3 models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.


Assuntos
Esôfago de Barrett/patologia , Neoplasias Esofágicas/epidemiologia , Fatores Etários , Esôfago de Barrett/fisiopatologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Inibidor p16 de Quinase Dependente de Ciclina , Progressão da Doença , Método Duplo-Cego , Endoscopia , Neoplasias Esofágicas/patologia , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Curva ROC , Reprodutibilidade dos Testes , Medição de Risco
17.
Bioinform Biol Insights ; 1: 127-136, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18425214

RESUMO

BACKGROUND AND AIMS: Because of the extremely low neoplastic progression rate in Barrett's esophagus, it is difficult to diagnose patients with concomitant adenocarcinoma early in their disease course. If biomarkers existed in normal squamous esophageal epithelium to identify patients with concomitant esophageal adenocarcinoma, potential applications would be far-reaching. The aim of the current study was to identify global gene expression patterns in normal esophageal epithelium capable of revealing simultaneous esophageal adenocarcinoma, even located remotely in the esophagus. METHODS: Tissues comprised normal esophageal epithelia from 9 patients with esophageal adenocarcinoma, 8 patients lacking esophageal adenocarcinoma or Barrett's, and 6 patients with Barrett's esophagus alone. cDNA microarrays were performed, and pattern recognition in each of these subgroups was achieved using shrunken nearest centroid predictors. RESULTS: Our method accurately discriminated normal esophageal epithelia of 8/8 patients without esophageal adenocarcinoma or Barrett's esophagus and of 6/6 patients with Barrett's esophagus alone from normal esophageal epithelia of 9/9 patients with Barrett's esophagus and concomitant esophageal adenocarcinoma. Moreover, we identified genes differentially expressed between the above subgroups. Thus, based on their corresponding normal esophageal epithelia alone, our method accurately diagnosed patients who had concomitant esophageal adenocarcinoma. CONCLUSIONS: These global gene expression patterns, along with individual genes culled from them, represent potential biomarkers for the early diagnosis of esophageal adenocarcinoma from normal esophageal epithelia. Genes discovered in normal esophagus that are differentially expressed in patients with vs. without esophageal adenocarcinoma merit further pursuit in molecular genetic, functional, and therapeutic interventional studies.

18.
Bioinform Biol Insights ; 1: 1-16, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18389087

RESUMO

We performed high-throughput cDNA sequencing in colorectal adenocarcinoma and matching normal colorectal epithelium. All six hundred three genes in the UCSC database that were expressed in colon cancers and contained open reading frames of 1000 nucleotides or less were selected for study (total basepairs/bp, 366,686). 304,350 of these 366,686 bp (83.0%) were amplified and sequenced successfully. Seventy-eight sequence variants present in germline (i.e. normal) as well as matching somatic (i.e. tumor) DNA were discovered, yielding a frequency of 1 variant per 3,902 bp. Fifty-one of these sequence variants were homozygous (26 synonymous, 25 non-synonymous), while 27 were heterozygous (11 synonymous, 16 non-synonymous). Cancer tissue contained only one sequence-altered allele of the gene ATP50, which was present heterozygously alongside the wild-type allele in matching normal epithelium. Despite this relatively large number of bp and genes sequenced, no somatic mutations unique to tumor were found. High-throughput cDNA sequencing is a practical approach for detecting novel sequence variations and alterations in human tumors, such as those of the colon.

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