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

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，癌细胞往往通过操纵DNA修复机制而对治疗产生抵抗力。相反，不断暴露在环境和内源性DNA损伤剂下的分裂细胞可能会因为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是至关重要的。在我们的初步研究中，我们发现了一类新的基因表达调控因子、microRNAs和DNA修复蛋白之间的新联系。MicroRNAs(MiRNAs)是一种小的非编码RNA，通常会抑制基因表达。越来越多的证据表明，miRNAs在癌细胞中错误表达。值得注意的是，异位过表达下调DNA修复蛋白的miRNAs可能会使癌细胞对辐射和其他遗传毒性试剂敏感。或者，缺失这些miRNAs的肿瘤可能会对传统的癌症治疗产生抗药性。DNA修复是一种“看家”功能，而微小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实验系统，我们鉴定了microRNAs(MiRNAs)，miR-24和一个多顺反子miRNA簇，包括miRNAs(183，96,182)，它们在终末分化的未分裂细胞中上调，但在分裂细胞中对电离辐射(IR)反应迅速下调。我们假设，在有丝分裂后细胞中，DNA损伤诱导细胞凋亡，而miRNAs减弱促进细胞死亡的DNA修复机制。相反，作为对IR的反应，miRNAs在分裂细胞时被下调，以增强DNA修复蛋白的产生，并增强DNA损伤反应。为了支持这一观点，我们观察到，miR-24下调了关键的DSB修复蛋白H2 AX的表达，并阻碍了终末分化血细胞中的DSB修复。我们计划研究的miRNAs(182,183和96)已经被注意到在各种肿瘤中异常表达。这些miRNAs对癌症的直接影响可能是通过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miRNAs-183，96和182的靶点。初步实验验证了miR-182调控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在转化细胞系和原代细胞中由miRNAs-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miRNAs对DSB修复的影响，并确定它们对每条修复途径的影响。最后，我们将评估表达这些miRNAs的癌细胞的放射敏感性。目前对miRNAs在DNA修复中的作用了解有限，本研究将解决这一问题，并阐明miRNAs对放射治疗的影响。

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.