Investigate role of microRNA cluster 183-96-182 in DNA repair and radiosensitivit

研究 microRNA 簇 183-96-182 在 DNA 修复和放射敏感性中的作用

• 批准号: 8434262
• 负责人: Dipanjan Chowdhury
• 金额: $ 33.11万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8434262
• 关键词: Address; Algorithms; Apoptosis; Attenuated; BRCA1 gene; Biochemical; Bioinformatics; Biological Assay; Blood Cells; Breast; Cancer Biology; Cell Death; Cell Differentiation process; Cell Line; Cells; Chromosome Breakage; Chronic Lymphocytic Leukemia; Clinical; Comet Assay; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA repair protein; Functional RNA; Gastrointestinal tract structure; Gene Expression; Gene Targeting; Goals; Hematopoietic; Housing; In Vitro; Interphase Cell; Ionizing radiation; Kidney; Lung; Malignant Neoplasms; Measures; Mediating; Messenger RNA; MicroRNAs; Mitotic; Molecular Profiling; Monitor; Neuraxis; Nonhomologous DNA End Joining; Normal Cell; Ovary; Pathway interactions; Phenotype; Production; Prostate; Proteins; Pulsed-Field Gel Electrophoresis; Radiation; Radiation Tolerance; Radiation therapy; Reagent; Reporter; Resistance; Resistance development; Role; Specificity; System; Transcript; Transformed Cell Line; attenuation; cancer cell; cancer therapy; chemotherapeutic agent; cytotoxic; homologous recombination; irradiation; melanoma; member; neoplastic cell; novel; outcome forecast; overexpression; public health relevance; repaired; research study; response; therapy resistant; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Radiation and chemotherapeutic agents eradicate tumors by inducing irreparable DNA damage. However, cancer cells often develop resistance to therapy by manipulating the DNA repair machinery. Conversely, a dividing cell constantly exposed to environmental and endogenous DNA damaging agents can transform into a tumor due to incorrect repair. Therefore the expression level of DNA repair proteins is critical both for cancer therapy and tumorigenesis. In our preliminary studies we have discovered a novel connection between a new class of gene expression regulators, microRNAs and DNA repair proteins. MicroRNAs (miRNAs) are small non-coding RNAs that typically dampen gene expression. There is accumulating evidence that miRNAs are mis-expressed in cancer cells. It is noteworthy that ectopic overexpression of miRNAs downregulating DNA repair proteins could sensitize cancer cells to radiation and other genotoxic reagents. Alternatively, tumors that delete these miRNAs may develop resistance to conventional cancer therapy. DNA repair is a 'house keeping' function, and micro-RNA mediated attenuation of DNA repair may appear counter intuitive. However, normal cells down modulate DNA repair in a terminally differentiated state where overall DNA repair is downregulated. Using the experimental system of in vitro hematopoietic cell differentiation, we have identified microRNAs (miRNAs), miR-24 and a polycistronic miRNA cluster including miRNAs (183, 96, 182), that are upregulated in terminally differentiated non-dividing cells but are rapidly down regulated in response to ionizing radiation (IR) in dividing cells. We hypothesize that in post-mitotic cells DNA damage induces apoptosis and miRNAs attenuate the DNA repair machinery promoting cell death. Conversely, in response to IR the miRNAs are downmodulated in dividing cells to accentuate the production of DNA repair proteins and boost the DNA damage response. In support of this contention, we observed, that miR-24, downregulates the expression of a key DSB repair protein, H2AX and impedes DSB repair in terminally differentiated blood cells. The miRNAs (182,183 and 96) that we propose to study have already been noted for aberrant expression in a variety of tumors. A direct effect of these miRNAs on cancer could be by dysregulation of the DNA repair machinery. Bioinformatic predictions suggest that several DNA repair genes, such as, BRCA1, ATR, XLF, etc. are targeted by the miRNAs-183, 96 and 182. Preliminary experiments validate the prediction that miR-182 regulates BRCA1. In Aim #1 we will use different computational, and biochemical strategies, to identify and validate DNA repair factors targeted by miRNAs-183, 96 and 182 in transformed cell lines and primary cells. In Aim #2 we will systematically study the effect of these miRNAs on DSB repair and determine their impact on each repair pathway. Finally we will evaluate the radiosensitivity of cancer cells expressing these miRNAs. There is limited understanding of the role of miRNAs in DNA repair and this study will address this issue and also elucidate the impact of miRNAs on radiotherapy.

描述（由申请人提供）： 放射和化疗药物通过诱导不可修复的 DNA 损伤来根除肿瘤。然而，癌细胞经常通过操纵 DNA 修复机制对治疗产生耐药性。相反，持续暴露于环境和内源性 DNA 损伤剂的分裂细胞可能会因不正确的修复而转化为肿瘤。因此，DNA 修复蛋白的表达水平对于癌症治疗和肿瘤发生都至关重要。在我们的初步研究中，我们发现了一类新型基因表达调节因子、microRNA 和 DNA 修复蛋白之间的新联系。 MicroRNA (miRNA) 是一种小型非编码 RNA，通常会抑制基因表达。越来越多的证据表明 miRNA 在癌细胞中错误表达。值得注意的是，下调 DNA 修复蛋白的 miRNA 异位过度表达可能会使癌细胞对辐射和其他基因毒性试剂敏感。或者，删除这些 miRNA 的肿瘤可能会对传统癌症治疗产生耐药性。 DNA 修复是一种“管家”功能，微小 RNA 介导的 DNA 修复减弱可能看起来与直觉相反。然而，正常细胞在终末分化状态下下调 DNA 修复，其中整体 DNA 修复下调。利用体外造血细胞分化的实验系统，我们鉴定了 microRNA (miRNA)、miR-24 和包括 miRNA (183, 96, 182) 在内的多顺反子 miRNA 簇，它们在终末分化的非分裂细胞中上调，但在分裂细胞中响应电离辐射 (IR) 快速下调。我们假设，在有丝分裂后细胞中，DNA 损伤会诱导细胞凋亡，而 miRNA 会减弱促进细胞死亡的 DNA 修复机制。相反，为了响应 IR，分裂细胞中的 miRNA 会下调，以增强 DNA 修复蛋白的产生并增强 DNA 损伤反应。为了支持这一论点，我们观察到 miR-24 下调关键 DSB 修复蛋白 H2AX 的表达，并阻碍终末分化血细胞中的 DSB 修复。我们建议研究的 miRNA（182,183 和 96）已被发现在多种肿瘤中存在异常表达。这些 miRNA 对癌症的直接影响可能是 DNA 修复机制的失调。生物信息学预测表明，几个DNA修复基因，如BRCA1、ATR、XLF等是miRNA-183、96和182的靶标。初步实验验证了miR-182调节BRCA1的预测。在目标#1中，我们将使用不同的计算和生化策略来识别和验证转化细胞系和原代细胞中 miRNA-183、96 和 182 靶向的 DNA 修复因子。在目标#2 中，我们将系统地研究这些 miRNA 对 DSB 修复的影响，并确定它们对每个修复途径的影响。最后我们将评估表达这些 miRNA 的癌细胞的放射敏感性。人们对 miRNA 在 DNA 修复中的作用的了解有限，本研究将解决这个问题并阐明 miRNA 对放射治疗的影响。