RESUMO
BACKGROUND: Human chromosome arm 3p is often affected in various epithelial tumors, and several tumor suppressor genes were recently identified in this region. The most affected is 3p21 region that is 50-100% rearranged in more than 30 types of malignancies, mostly in epithelial cancers: lung, breast, ovarian, cervical, kidney, head and neck, nasopharyngeal, colon etc. These cancers are responsible for 90% of cancer deaths. AIM: To perform the detailed analysis of 3p (especially 3p21 region) to discover novel potential oncogenes and/or tumor suppressors. METHODS: To find novel "hot spots" and genes involved in major cancers, dense 3p microsatellite markers (altogether 24 ) were allelotyped in four epithelial carcinomas (272 patients in total): breast (BC), renal cell (RCC), non-small cell lung (NSCLC) and epithelial ovarian (EOC) cancers. RESULTS: As a main result, a novel region, frequently affected in BC, RCC, NSCLC and EOC was localized between markers D3S2409 and D3S3667 in the 3p21.3. This region (MECA3, major epithelial cancers affected region No. 3) covers numerous UniGene clusters, including genes involved in vital cell functions and carcinogenesis (e.g. MST1, MSTR1/RON, GPX1 and RHOA). The homozygous deletions were detected in the GPX1 in RCC (12%, 6 of 50 cases) and BC (1 of 37 cases). At the same time, amplifications and multiplications within the RHOA putative oncogene were identified in BC and RCC. CONCLUSIONS: The data suggest that genes with potential oncogenic features are located in the close proximity to putative tumor suppressor gene(s) (TSG(s)) in the MECA3. Multiplication of the RHOA was not reported before. Significant correlation of allelic alterations in the, AP20, MECA3 and LUCA regions with tumor progression was found for some common histological tumor subtypes (e.g. clear cell RCC, and serous EOC).
Assuntos
Cromossomos Humanos Par 3/genética , Genes Supressores de Tumor , Oncogenes , Desequilíbrio Alélico/genética , Deleção Cromossômica , Progressão da Doença , Amplificação de Genes/genética , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica , Glutationa Peroxidase/genética , Homozigoto , Humanos , Repetições de Microssatélites/genética , Neoplasias/genética , Polimorfismo Genético , Proteína rhoA de Ligação ao GTP/genética , Glutationa Peroxidase GPX1RESUMO
UNLABELLED: Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. AIM: To validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene in human RCC samples. METHODS: DNA and RNA were isolated from freshly-frozen renal tumors' samples (n = 12) and from normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequencing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative quantification 2-(DeltaDeltaCP) method was used. Nonparametric tests (Wilcoxon, Kruskal - Wallis and Mann - Whitney) were applied for statistical data analysis using the BioStat software. RESULTS: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of 12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age. Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio (< 0.85 and >or= 0.35), thus were considered as hemizygous deletions. 3 samples showed ratio (> 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes in methylation status of NKIRAS1 were found in RCC. CONCLUSIONS: We have validated the results of NotI microarray analysis of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor.
