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Liming Zhang,
Tan Deng,
Xiayu Li,
Huaying Liu,
Houde Zhou,
Jian Ma,
Minghua Wu,
Ming Zhou,
Shourong Shen,
Xiaoling Li, Zhaoxia Niu,
Wenling Zhang,
Lei Shi,
Bo Xiang,
Jianhong Lu,
Li Wang,
Dan Li,
Hailin Tang,
Guiyuan Li
[show abstract]
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ABSTRACT: microRNAs (miRNAs) are small non-coding RNAs and have been implicated in the pathology of various diseases, including cancer. Here we report that the miRNA profiles have been changed after knockdown of one of the most important oncogene c-MYC or re-expression of a candidate tumor suppressor gene SPLUNC1 in nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown and SPLUNC1 re-expression can down-regulate microRNA-141 (miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of miR-141 could affect cell cycle, apoptosis, cell growth, migration and invasion in NPC cells. We found that BRD3, UBAP1 and PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting. BRD3 and UBAP1 are both involved in NPC carcinogenesis as confirmed through our previous studies and PTEN is a crucial tumor suppressor in many tumor types. BRD3 is involved in the regulation of the Rb/E2F pathway. Inhibition of miR-141 could affect some important molecules in the Rb/E2F, JNK2 and AKT pathways. It is well known that carcinogenesis of NPC is involved in the networks of genetic and epigenetic alteration events. We propose that miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3, UBAP1 and PTEN may constitute a gene-miRNA network to contribute to NPC development.
Carcinogenesis 04/2010; 31(4):559-66. · 5.70 Impact Factor
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Huaying Liu,
Ming Zhou,
Xiaomin Luo,
Liming Zhang, Zhaoxia Niu,
Cong Peng,
Jian Ma,
Shuping Peng,
Houde Zhou,
Bo Xiang,
Xiayu Li,
Shufang Li,
Jiajin He,
Xiaoling Li,
Guiyuan Li
[show abstract]
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ABSTRACT: Bromodomain is an evolutionally conserved domain that is found in proteins strongly implicated in signal-dependent transcriptional regulation. Genetic alterations of bromodomain genes contributed to the development of many human cancers and other disorders. BRD7 is a recently identified bromodomain gene. It plays a critical role in cellular growth, cell cycle progression, and signal-dependent gene expression. Previous studies showed that BRD7 gene exhibited much higher-level of mRNA expression in normal nasopharyngeal epithelia than in nasopharyngeal carcinoma (NPC) biopsies and cell lines. However, little is known about its transcriptional regulation. In this study, we explored the transcriptional regulation of BRD7 gene.
Potential binding sites of transcription factors within the promoter region of BRD7 gene were predicted with MatInspector Professional http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl. Mutation construct methods and luciferase assays were performed to define the minimal promoter of BRD7 gene. RT-PCR and western blot assays were used to detect the endogenous expression of transcription factor Sp1, c-Myc and E2F6 in all cell lines used in this study. Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) were used to detect the direct transcription factors that are responsible for the promoter activity of BRD7 gene. DNA vector-based siRNA technology and cell transfection methods were employed to establish clone pools that stably expresses SiRNA against c-Myc expression in nasopharyngeal carcinoma 5-8F cells. Real-time PCR was used to detect mRNA expression of BRD7 gene in 5-8F/Si-c-Myc cells.
We defined the minimal promoter of BRD7 gene in a 55-bp region (from -266 to -212bp), and identified that its promoter activity is inversely related to c-Myc expression. Sp1 binds to the Sp1/Myc-Max overlapping site of BRD7 minimal promoter, and slightly positively regulate its promoter activity. c-Myc binds to this Sp1/Myc-Max overlapping site as well, and negatively regulates the promoter activity and endogenous mRNA expression of BRD7 gene. Knock-down of c-Myc increases the promoter activity and mRNA level of BRD7 gene. The luciferase activity of the mutated promoter constructs showed that Sp1/Myc-Max overlapping site is a positive regulation element of BRD7 promoter.
These studies provide for the first time the evidence that c-Myc is indeed a negative regulator of BRD7 gene. These findings will help to further understand and uncover the bio-functions of BRD7 gene involved in the pathogenesis of NPC.
BMC Molecular Biology 01/2009; 9:111. · 2.86 Impact Factor
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[show abstract]
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ABSTRACT: Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription.
The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.
We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals.
BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.
BMC Cancer 10/2008; 8:253. · 3.01 Impact Factor
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Huaying Liu,
Xiaoling Li, Zhaoxia Niu,
Liming Zhang,
Ming Zhou,
He Huang,
Jiajin He,
Wenling Zhang,
Lan Xiao,
Yunlian Tang,
Li Wang,
Guiyuan Li
[show abstract]
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ABSTRACT: BRD7 is a novel bromodomain gene. It plays critical role in cell growth, cell cycle progression, and signal-dependent gene expression. Overexpression of the BRD7 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and cell cycle progression from G1 to S phase. However, little is known about its bio-functions because of the unavailability of a specific BRD7 antibody. In this study, for the first time, we generated a highly specific BRD7 antibody. It is able to specifically recognize recombinant GST-BRD7N protein with a molecular mass of 65 kDa and recognize BRD7-Myc and endogenously expressed BRD7 protein with an approximate molecular mass of 75 kDa, which corresponds well with the calculated molecular mass of the BRD7 protein. More importantly, with these antisera, we analyzed BRD7 distribution in the human fetus by Western blot and immunohistochemistry assays. Obvious nuclear expression of BRD7 protein presents in human cerebellum, pancreas, intestines, liver, and kidney. Cardiomyocyte shows high cytoplasm expression of the BRD7 protein. Weak nuclear expression of the BRD7 protein is found in human cerebrum, lung, and stomach. These data may help to further study the cellular role of the BRD7 gene. In particular, the prepared BRD7 antibody will be helpful for studying the bio-functions of endogenously expressed BRD7 protein.
Journal of Histochemistry and Cytochemistry 07/2008; 56(6):531-8. · 2.72 Impact Factor
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Huaying Liu,
Cong Peng,
Ming Zhou,
Jie Zhou,
Shourong Shen,
Houde Zhou,
Wei Xiong,
Xiaomin Luo,
Shuping Peng, Zhaoxia Niu,
Jue Ouyang,
Xiaoling Li,
Guiyuan Li
[show abstract]
[hide abstract]
ABSTRACT: BRD7, a novel bromodomain gene, encodes a protein that inhibits cell growth and cell cycle progression by transcriptional regulation of some cell cycle-related genes. Its transcriptional down-expression has been shown to be critical to the pathogenesis of Nasopharyngeal carcinoma (NPC). Little is known about the transcriptional mechanisms controlling BRD7 gene expression. In this paper, we have characterized the 5' regulatory region of the BRD7 gene in order to understand the molecular mechanisms regulating its expression. Transient transfection results suggested that the analyzed upstream sequences of the BRD7 gene might contain some important but not sufficient sequence information to confer the cell-type specificity of BRD7 gene expression. Further analysis with a series of deletions demonstrated that a 125-bp region was required for the basal promoter activity of the BRD7 gene. Results from ChIP and EMSA indicated that the promoter was responsive to Sp1, E2F, and E2F6. All of these suggest a possible mechanism that transcriptional factor Sp1, E2F, and E2F-6 are associated in the BRD7 promoter region and regulate BRD7 promoter activity. Taken together, these results will help to better understand the role of the BRD7 gene in signal-dependent transcriptional regulation, and to develop new reagents for therapeutic upregulation of the BRD7 gene in NPC.
DNA and Cell Biology 07/2006; 25(6):346-58. · 2.07 Impact Factor
