RESUMO
In this study, our purpose is to clarify the role of specificity protein 1 (Sp1) in transcription activation of the four and half lim 2 (FHL2). pLuc595 which contained -1163nt to -568nt upstream ATG starting codon displayed the highest while pLuc382 (-950nt to -568nt) displayed lowest transcription activities in GI cancer cells. Meanwhile, suppression of Sp1 by siRNA or chemical inhibitor, mithramycin A (MIT) inhibited the promoter activity and FHL2 expression. Bioinformatics analysis showed two putative Sp1 binding elements within pLuc595 while EMSA assay demonstrated that the wild type but not the mutated probe containing the distal element bound to the cell nuclear protein specifically. Mutation of the distal Sp1 binding sequence suppressed the transcription activity of pLuc595. In vivo study showed that both Sp1 and FHL2 were highly expressed by cancer cells but not the normal GI tissues with their expressions being positive correlated. These data identified a functional Sp1 positively regulatory element (-1058nt to -1049nt) within FHL2 promoter, and the positive correlation between Sp1 and FHL2 in expressing pattern and signal mechanism strongly suggested their synergized effect in carcinogenesis and progression of GI cancers.
Assuntos
Regulação da Expressão Gênica , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Neoplasias Gastrointestinais/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
It has been reported that XAF1 expression in gastric cancer is negatively correlated with p53. Our purpose was to clarify the regulatory mechanism of p53 on XAF1 expression. The effects of overexpressed wild-type and mutant p53 on XAF1 expression were evaluated. Binding capacity of core XAF1 promoter sequence to the recombinant p53 protein was examined. Site-directed mutation of putative p53 binding sequence and p53 knockdown by siRNA were performed. The protein expression and promoter activities of XAF1 in cells with null p53 were higher than that with wild-type and mutant p53. Ectopic overexpression of wild-type p53 suppressed XAF1 expression. A half-site (-95 to -86 nt) and a quarter-site (-4 to +1 nt) of p53 responsive element were found within XAF1 promoter. Both sequences bound to recombinant p53 effectively and specifically. Site-mutation of p53 responsive sequences abrogated the binding capacity. However, only the mutation of half-site increased XAF1 promoter activities. Suppression of p53 not only decreased the binding capacity of p53 responsive halfsite but also increased XAF1 transcription. In conclusion, we demonstrated that p53 could suppress the transcription of XAF1 through interaction with a high affinity responsive element (-95 to -86 nt) within XAF1 promoter, indicating a novel exclusive mechanism between these two tumor suppressors.
Assuntos
Neoplasias do Colo/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Proteína Supressora de Tumor p53/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Proteínas de Neoplasias/metabolismo , Interferência de RNA , Proteínas Recombinantes/metabolismo , Elementos de Resposta , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: To clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods. METHODS: With the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method. RESULTS: A 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes. CONCLUSION: The prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.
