RESUMO
Prostate cancer incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to prostate cancer health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA prostate cancer. A comprehensive approach of using cell lines, tissue samples, and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR array, qRT-PCR, cell cycle, luciferase reporter, immunoblot, and IHC. Various statistical approaches such as Kaplan-Meier, uni-, and multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell-cycle pathway activation in AA patients with prostate cancer. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion, and induced cell-cycle arrest. Inhibition of miR-130b modulated critical prostate cancer-related biological pathways in AA compared with EA prostate cancer patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA prostate cancer. miR-130b is a significant contributor to prostate cancer racial disparity as its overexpression is a risk factor for poor prognosis in AA patients with prostate cancer. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of prostate cancer in AA patients.
Assuntos
Disparidades nos Níveis de Saúde , MicroRNAs/genética , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/etnologia , Neoplasias da Próstata/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/etnologia , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Negro ou Afro-Americano , Idoso , Idoso de 80 Anos ou mais , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Células PC-3 , Prognóstico , Neoplasias da Próstata/mortalidade , Fatores Raciais , Fatores de Risco , Análise de Sobrevida , Células Tumorais Cultivadas , Regulação para Cima/genéticaRESUMO
The molecular heterogeneity of clear cell renal carcinoma (ccRCC) makes prediction of disease progression and therapeutic response difficult. Thus, this report investigates the functional significance, mechanisms of action, and clinical utility of miR-182-5p and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1/NEAT2), a long noncoding RNA (lncRNA), in the regulation of kidney cancer using human kidney cancer tissues as well as in vitro and in vivo model systems. Profiling of miR-182-5p and MALAT-1 in human renal cancer cells and clinical specimens was done by quantitative real-time PCR (qPCR). The biological significance was determined by series of in vitro and in vivo experiments. The interaction between miR-182-5p and MALAT-1 was investigated using luciferase reporter assays. In addition, the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell-cycle progression were assessed in ccRCC cells. The data indicate that miR-182-5p is downregulated in ccRCC; the mechanism being CpG hypermethylation as observed from 5-Aza CdR treatment that decreased promoter methylation and expression of key methylation regulatory genes like DNMT1, DNMT3a, and DNMT3b Overexpression of miR-182-5p-inhibited cell proliferation, colony formation, apoptosis, and led to G2-M-phase cell-cycle arrest by directly targeting MALAT-1 Downregulation of MALAT-1 led to upregulation of p53, downregulation of CDC20, AURKA, drivers of the cell-cycle mitotic phase. Transient knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Finally, overexpression of miR-182-5p decreased tumor growth in mice, compared with controls; thus, demonstrating its antitumor effect in vivo Implications: This is the first study that offers new insight into role of miR-182-5p/MALAT-1 interaction on inhibition of ccRCC progression. Mol Cancer Res; 16(11); 1750-60. ©2018 AACR.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/biossíntese , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Masculino , Camundongos , Camundongos Nus , MicroRNAs/administração & dosagem , MicroRNAs/biossíntese , MicroRNAs/genética , Mitose/fisiologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study aims to investigate the role of miR-203-HOTAIR interaction in the suppression of renal cell carcinoma (RCC). We employed series of in vitro assays such as proliferation, invasion, migration, and colony formation along with in vivo tumor xenograft model. Profiling of miR-203 and HOTAIR expression revealed that miR-203 was significantly underexpressed, whereas HOTAIR was overexpressed in RCC cell lines and clinical specimens compared with normal cell line and tissue. Both miR-203 and HOTAIR expression significantly distinguished malignant from normal tissues and significantly correlated with clinicopathologic characteristics of patients. Overexpression of miR-203 significantly inhibited proliferation, migration, and invasion with an induction of apoptosis and cell-cycle arrest. However, HOTAIR suppression resulted in the similar functional effects in the same RCC cell lines. In silico, RNA-22 algorithm showed a binding site for miR-203 in HOTAIR. We observed a direct interaction between miR-203 and HOTAIR by RNA-immunoprecipitation (RIP) and luciferase reporter assays. We show that miR-203-HOTAIR interaction resulted in the inhibition of epithelial-to-mesenchymal transition (EMT) and metastatic genes as indicated by induction of key metastasis-suppressing proteins E-cadherin, claudin (epithelial markers), and PTEN along with induction of tumor suppressor genes p21 and p27. A significant decrease in vimentin (mesenchymal marker), KLF4, and Nanog (stemness markers) was also observed. This is the first report demonstrating miR-203-mediated regulation of HOTAIR induces tumor suppressor effects in RCC by regulating EMT and metastatic pathway genes. Thus, the study suggests that therapeutic regulation of HOTAIR by miR-203 overexpression may provide an opportunity to regulate RCC growth and metastasis. Mol Cancer Ther; 17(5); 1061-9. ©2018 AACR.
Assuntos
Carcinogênese/genética , Carcinoma de Células Renais/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Renais/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Apoptose/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Fator 4 Semelhante a Kruppel , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The most frequent alteration in the prostate oncogenome is loss of chromosome (chr) 8p21 that has been associated with loss of NKX3.1 homeobox gene. Chr8p21 deletions increase significantly with tumor grade and are associated with poor prognosis in prostate cancer (PCa), suggesting critical involvement of this region in tumor progression. Recent studies suggest that apart from NKX3.1, this region harbors alternative tumor suppressors that are yet undefined. We proposed a novel, paradigm shifting hypothesis that this locus is associated with a miRNA gene cluster-miR-3622a/b- that plays a crucial suppressive role in PCa. Here we demonstrate the crucial role of miR-3622a in prostate cancer epithelial-to-mesenchymal transition (EMT). MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a expression is widely downregulated and is significantly correlated with poor survival outcome and tumor progression. To understand the functional significance of miR-3622a, knockdown and overexpression was performed using non-transformed prostate epithelial and PCa cell lines, respectively, followed by functional assays. Our data demonstrate that endogenous miR-3622a expression is vital to maintain the epithelial state of normal and untransformed prostate cells. miR-3622a expression inhibits EMT, progression and metastasis of PCa in vitro and in vivo. Further, we found that miR-3622a directly targets EMT effectors ZEB1 and SNAI2. In view of these data, we propose that frequent loss of miR-3622a at chr8p21 region leads to induction of EMT states that in turn, promotes PCa progression and metastasis. This study has potentially significant implications in the field of prostate cancer as it identifies an important miRNA component of a frequently lost chromosomal region with critical roles in prostate carcinogenesis which is a highly significant step towards understanding the mechanistic involvement of this locus. Also, our study indicates that miR-3622a is a novel PCa biomarker and potential drug target for developing therapeutic regimens against advanced PCa.
Assuntos
Transição Epitelial-Mesenquimal/genética , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromossomos Humanos Par 8/genética , Metilação de DNA/genética , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genoma Humano , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
The proteoglycan versican (VCAN) promotes tumor progression and enhances metastasis in several cancers; however, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Recent evidence suggests that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC. Thus, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. VCAN expression was analyzed using three RCC and normal kidney cell lines as well as a clinical cohort of 84 matched ccRCC and normal renal tissues. Functional analyses on growth and progression properties were performed using VCAN-depleted ccRCC cells. Microarray expression profiling was employed to investigate the target genes and biologic pathways involved in VCAN-mediated ccRCC carcinogenesis. ccRCC had elevated VCAN expression in comparison with normal kidney in both cell lines and clinical specimens. The elevated expression of VCAN was significantly correlated with metastasis (P < 0.001) and worse 5-year overall survival after radical nephrectomy (P = 0.014). In vitro, VCAN knockdown significantly decreased cell proliferation and increased apoptosis in Caki-2 and 786-O cells, and this was associated with alteration of several TNF signaling-related genes such as TNFα, BID, and BAK Furthermore, VCAN depletion markedly decreased cell migration and invasion which correlated with reduction of MMP7 and CXCR4. These results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis and thus is an attractive target for novel diagnostic, prognostic, and therapeutic strategies.Implications: This study highlights the oncogenic role of VCAN in renal cell carcinogenesis and suggests that this gene has therapeutic and/or biomarker potential for renal cell cancer. Mol Cancer Res; 15(7); 884-95. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Proteínas de Neoplasias/genética , Versicanas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Análise Serial de TecidosRESUMO
The incidence of prostate cancer (PCa) among African-Americans (AfA) is significantly higher than Caucasian-Americans (CaA) but the genetic basis for this disparity is not known. To address this problem, we analyzed miRNA expression in AfA (n = 81) and CaA (n = 51) PCa patients. Here, we found that miR-24 is differentially expressed in AfA and CaA PCa patients and attempt to clarify its role in AfA patients. Also, the public sequencing data of the miR-24 promoter confirmed that it was highly methylated and down-regulated in PCa patients. Utilizing a VAMCSF and NDRI patient cohorts, we discovered that miR-24 expression was linked to a racial difference between AfA/CaA PCa patients. Interestingly, miR-24 was restored after treatment of PCa cells with 5Aza-CdR in an AfA cell line (MDA-PCa-2b), while restoration of miR-24 was not observed in CaA cells, DU-145. Ectopic expression of miR-24 showed decreased growth and induced apoptosis, though the effect was less in the CaA cell line compared to the AfA cell line. Finally, we found unique changes in biological pathways and processes associated with miR-24 transfected AfA cells by quantitative PCR-based gene expression array. Evaluation of the altered pathways showed that AR, IGF1, IGFBP5 and ETV1 were markedly decreased in the AfA derived cell line compared with CaA cells, and there was a reciprocal regulatory relationship of miR-24/target expression in prostate cancer patients. These results demonstrate that miR-24 may be a central regulator of key events that contribute to race-related tumorigenesis and has potential to be a therapeutic agent for PCa treatment.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Humanos , Incidência , Masculino , MicroRNAs/biossíntese , Neoplasias da Próstata/etnologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3' untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans.
Assuntos
Negro ou Afro-Americano/genética , MicroRNAs/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , População Branca/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Deleção Cromossômica , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Neoplasias da Próstata/etnologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Transdução de Sinais/genética , Regiões 3' não Traduzidas/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Metilação de DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transplante HeterólogoRESUMO
To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.
RESUMO
Genomic loss of chromosome (chr) 8p21 region, containing prostate-specific NKX3.1 gene, is a frequent alteration of the prostate cancer (PCa) oncogenome. We propose a novel, paradigm shifting hypothesis that this frequently deleted locus is also associated with a cluster of microRNA genes- miR-3622a/b- that are lost in PCa and play an important mechanistic role in progression and metastasis. In this study, we demonstrate the role of miR-3622b in prostate cancer. Expression analyses in a cohort of PCa clinical specimens and cell lines show that miR-3622b expression is frequently lost in prostate cancer. Low miR-3622b expression was found to be associated with tumor progression and poor biochemical recurrence-free survival. Further, our analyses suggest that miR-3622b expression is a promising prostate cancer diagnostic biomarker that exhibits 100% specificity and 66% sensitivity. Restoration of miR-3622b expression in PCa cell lines led to reduced cellular viability, proliferation, invasiveness, migration and increased apoptosis. miR-3622b overexpression in vivo induced regression of established prostate tumor xenografts pointing to its therapeutic potential. Further, we found that miR-3622b directly represses Epidermal Growth Factor Receptor (EGFR). In conclusion, our study suggests that miR-3622b plays a tumor suppressive role and is frequently downregulated in prostate cancer, leading to EGFR upregulation. Importantly, miR-3622b has associated diagnostic, prognostic and therapeutic potential. Considering the association of chr8p21 loss with poor prognosis, our findings are highly significant and support a novel concept that associates a long standing observation of frequent loss of a chromosomal region with a novel miRNA in prostate cancer.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 8 , Receptores ErbB/genética , Genes Supressores de Tumor/fisiologia , MicroRNAs/fisiologia , Neoplasias da Próstata/patologia , Idoso , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidadeRESUMO
Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2'-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Metilação de DNA , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Fumar Tabaco/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Apoptose , Azacitidina/análogos & derivados , Azacitidina/química , Linhagem Celular Tumoral , Ilhas de CpG , Citocromo P-450 CYP1A1/genética , Decitabina , Elementos Facilitadores Genéticos , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Hiperplasia Prostática/genética , Neoplasias da Próstata/genética , Sulfitos/química , Análise Serial de Tecidos , Xenobióticos/químicaRESUMO
BACKGROUND: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. METHODS: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses. RESULTS: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples. CONCLUSIONS: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.
Assuntos
Carcinoma de Células Renais/genética , Proteínas Cdc20/genética , Citocromo P-450 CYP1B1/genética , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Renais/genética , Apoptose , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proteínas Cdc20/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Citocromo P-450 CYP1B1/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Regulação para CimaRESUMO
A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).
Assuntos
MicroRNAs/biossíntese , Neoplasias da Próstata/genética , Humanos , Hibridização In Situ/métodos , Masculino , MicroRNAs/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Inclusão em Parafina , Prognóstico , Neoplasias da Próstata/metabolismo , Sondas RNA/química , Sondas RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fixação de TecidosRESUMO
DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Apoptose/genética , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Adenosina Trifosfatases/genética , Proteínas Adaptadoras de Sinalização CARD , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas do Citoesqueleto/biossíntese , DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Endonuclease PMS2 de Reparo de Erro de Pareamento , Invasividade Neoplásica/genética , Próstata , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Interferência de RNA , RNA Interferente PequenoRESUMO
We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21(WAF1/CIP1) and p27(KIP1) expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2'-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.
Assuntos
Proteína Morfogenética Óssea 2/genética , Carcinoma de Células Renais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Proteínas de Neoplasias/genética , Idoso , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína Morfogenética Óssea 2/biossíntese , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/cirurgia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes cdc , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/cirurgia , Túbulos Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Nefrectomia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Resultado do TratamentoRESUMO
OBJECTIVE: To study the methylation level in the promoter of caspase 8 associated protein 2 (CASP8AP2) gene between samples at diagnosis and in complete remission, and to investigate its relationship with clinical features and prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: Diagnostic DNA samples from 109 newly diagnosed children with ALL admitted from August 2007 to March 2010, and 94 ALL children in CR (complete remission) among them were collected. Bisulfite modification and MethyLight method established by our research team were used to determine the methylation level of the two key CpG sites (at -1189 and -1176) of the promoter of CASP8AP2 gene. RESULTS: The average methylation level of the two CpG sites in newly diagnosted samples was higher than that in CR samples (71.1% ± 1.7% vs 64.2% ± 21.2%) (P = 0.008). Analysis with receiver operating characteristic (ROC) curve showed that the area under curve was 0.687 (P = 0.024), indicating that the methylation level of the two CpG sites was able to predict relapse efficiently to some extent, 76.9% was chosed as a cutoff value to divide the patients into high methylation group (49 patients) and low methylation group (60 patients). The incidence of relapse in high methylation group was higher than that in low methylation group (20.4% vs 6.7%) (P = 0.044), five year relapse free survival in high methylation group was also lower than that in low methylation group (Log rank, P = 0.033). Furthermore, high methylation at new diagnosis were correlated with high level of minimal residual disease (MRD) before consolidation therapy (P = 0.011). In the 34 children with MRD ≥ 10(-4) at the end of induction remission, the relapse rate of high methylation patients was significantly higher than that of low methylation patients (8/16 vs 3/18)(P = 0.038). CONCLUSION: The abnormal hypermethylation of the two CpG sites (at -1189 and -1176) of the promoter of the CASP8AP2 gene is possibly associated with leukemogenesis in childhood ALL. The treatment outcome is more poor in patients with hypermethylation than that in patients with low methylation. The combination of the methylation level of the two CpG sites and MRD level at the end induction remission is able to predict relapse more effectively.
Assuntos
Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Reguladoras de Apoptose , Proteínas de Ligação ao Cálcio , Criança , Humanos , Neoplasia Residual , Prognóstico , Regiões Promotoras Genéticas , Recidiva , Indução de RemissãoRESUMO
Recently, long noncoding RNAs (lncRNA) have emerged as new gene regulators and prognostic markers in several cancers, including renal cell carcinoma (RCC). In this study, we investigated the contributions of the lncRNA MALAT1 in RCC with a specific focus on its transcriptional regulation and its interactions with Ezh2 and miR-205. We found that MALAT1 expression was higher in human RCC tissues, where it was associated with reduced patient survival. MALAT1 silencing decreased RCC cell proliferation and invasion and increased apoptosis. Mechanistic investigations showed that MALAT1 was transcriptionally activated by c-Fos and that it interacted with Ezh2. After MALAT1 silencing, E-cadherin expression was increased, whereas ß-catenin expression was decreased through Ezh2. Reciprocal interaction between MALAT1 and miR-205 was also observed. Lastly, MALAT1 bound Ezh2 and oncogenesis facilitated by MALAT1 was inhibited by Ezh2 depletion, thereby blocking epithelial-mesenchymal transition via E-cadherin recovery and ß-catenin downregulation. Overall, our findings illuminate how overexpression of MALAT1 confers an oncogenic function in RCC that may offer a novel theranostic marker in this disease.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/fisiologia , Adulto , Idoso , Sequência de Bases , Sítios de Ligação , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/metabolismo , Neoplasias Renais/patologia , Masculino , MicroRNAs , Pessoa de Meia-Idade , Invasividade Neoplásica , Complexo Repressor Polycomb 2/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Ativação TranscricionalRESUMO
ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas de Ligação ao Cálcio/biossíntese , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Criança , Pré-Escolar , China , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Valor Preditivo dos Testes , Recidiva , Taxa de SobrevidaRESUMO
Little is known about DNMT3A mutations in childhood acute lymphoblastic leukemia (ALL). We screened for DNMT3A mutations in exon 23 and its adjacent intron regions in diagnostic samples of 201 children with ALL. The cDNA samples from 82 patients were also sequenced to identify other mutations in the entire coding region. DNMT3A mutations were detected in exon 23 and its adjacent intron regions only in five patients (2.5%). There was only one mutation in exon 23 in two patients, respectively. In the other three patients, five intronic mutations were found. None of the mutations was found in the five corresponding complete remission samples. DNMT3A mutations were correlated with higher minimal residual disease at the end of remission induction (p = 0.078). Treatment outcome was obviously worse in patients with DNMT3A mutations than in other patients (p < 0.05). Thus, DNMT3A mutations can be found in a few children with ALL, and may have an adverse impact on prognosis.