Mutation Detection And Molecular Mechanism Research Of Tumor Related Genes

Part one:Mutation detection and LOVD Database for BRCA1/2Gene in the Chinese PopulationObjective:To created a novel variants database for BRCAI and BRCA2in the Chinese population using the Leiden Open Variation Database (LOVD) format, as part of the International Variome Project (HVP-China)Methods:We searched PubMed and some Chinese searching engines to collect all the variants of BRCAI and BRCA2in the Chinese population that have been reported. In addition, peripheral blood samples of the patients with familiar or early-onset breast cancer were colleted. DNA was abstracted, PCR and sequencing were used for detection of BRCAI gene. The novel variation observed in the gene test was also submitted to our LOVD database.Results:To date, our database includes136unique variants of BRCAI and62unique variants of BRCA2. There are some differences in BRCA1/2gene mutations between Chinese population and that of other ethnicities. Of the14pathogenic mutations reported most frequently,50%cannot be found in the Breast Cancer Information Core database.Conclusion:we have created the first known variation database of BRCA1/2in Chinese population. There are some differences in BRCA1/2gene mutations between Chinese population and that of other ethnicities. This database will provide a great convenience to researchers and clinicians who study, test, and diagnose Hereditary breast cancer and ovarian cancer (HBOC) and other cancers caused by mutations on BRCAI and BRCA2. This database can be of especial use for facilitating future genetic tests of these two genes in Chinese population.Part two:Function and Regulation of DTWD1Gene in Gastric CancerObjective:To establish the function of DTWD1, as well as the potential regulation factor and mechanism of this gene in gastric cancer cells.Methods:DTWD1expression level was detected by quantitative real-time RT-PCR in gastric cancer cell lines with or without TSA treatment, normal gastric cell line (GES), and normal or tumor samples from clinical surgery. The cells were transfected with different HDAC siRNAs to determine which HDAC contributes most to DTWD1dysregulation. Luciferase assay was used to determine the promoter region of DTWD1gene and ChIP was used to examine the binding of p53protein and DTWD1promoter. To assess the tumor-suppressor function of DTWD1, MTS assay and colony formation assay were carried out in cells transfected with DTWD1expression vector or siRNA. Western blot was used to detect protein expression level.Results: DTWD1is downregulated in gastric cancer and can be upregulated by TSA treatment. HDAC3is the most important HDAC which contributes to DTWD1down regulation in gastric cancer cell lines.The protein p53can bind to the promoter of DTWD1and p53is a positive regulator of DTWD1. HDAC3impacts the binding of p53protein and DTWDlgene. Ectopic DTWDl expression inhibits tumor cell growth and colony formation ability.Conclusion:DTWDl is a novel gene which functions as a tumor suppressor in gastric cancer. DTWDl was regulated through HDAC3repression and p53activation.