NDRG2诱导miR-630促进宫颈癌细胞辐射抗性的作用与机制

The mechanisms underlying radioresistance of cervical cancer remain to be elucidated. Our study supported by a grant from National Natural Science Foundation of China revealed that hypoxia and radiation induced NDRG2 expression in human cervical cancer cells in a HIF-1-dependent manner and that NDRG2 promoted radioresistance of cervical cancer cells through inhibiting radiation-induced apoptosis. We further demonstrated that miR-630 was up-regulated in NDRG2-overexpressed cells and radioresistant cervical cancer cells, miR-630 mimics promoted radioresistance while its inhibitor enhanced radiosensitivity of cervical cancer cells, and that NDRG2 promoted radiation-induced up-regulation of miR-630. Based on these data, we hypothesized that NDRG2 induced miR-630 expression and miR-630 in turn promoted radioresistance of cervical cancer cells. In the current study, reporter gene assay, EMSA and Chip assay will be employed to uncover how NDRG2 induced miR-630 expression. RIP-chip and iTRAQ will be used to identify miR-630 targets. miR-630 and its targets will be modulated in cervical cancer cells by infection or transfection of expressing vectors or antagomirs. Then the influence of miR-630 and its targets on radiosensitivity and DNA damage signal transduction will be explored. The study will deepen our understanding of tumor radioresistance and help us to find novel therapeutic targets for radio-sensitizing reagents.

宫颈癌辐射抗性的机制仍未完全阐明。申请者前一个已结题的国科金研究表明，乏氧和辐射通过HIF-1诱导NDRG2在宫颈癌细胞的表达，NDRG2通过抑制辐射诱导的凋亡而增强癌细胞的辐射抗性。进一步研究发现，miR-630在NDRG2过表达细胞及辐射抗性细胞中表达上调，其模拟物促进辐射抗性而抑制剂增强辐射敏感性，NDRG2促进辐射对miR-630的表达诱导作用。我们据此提出了NDRG2诱导miR-630促进宫颈癌细胞辐射抗性的理论假设。本项目将借助报告基因实验、EMSA、Chip明确NDRG2促进miR-630表达的机制，联用RIP-chip和iTRAQ技术寻找miR-630的靶分子，通过基因转染和体内外实验阐明miR-630及其靶分子对辐射敏感性的调控作用，明确它们对辐射诱导的DNA损伤信号通路的影响。研究结果有助于加深对肿瘤辐射抗性机制的理解，为寻找新的具有辐射增敏作用的治疗靶点提供理论依据。

我们前期研究发现miR-630 在 NDRG2 过表达细胞及辐射抗性细胞中表达上调。本课题旨在探讨辐射诱导 miR-630 促进宫颈癌细胞辐射抗性的作用及分子机制。.取得的研究结果有：1.辐射可诱导宫颈癌Hela细胞中miR-630表达上调，并具有剂量和时间依赖性；2. miR-630可增强辐射后宫颈癌Hela细胞的活性及克隆形成能力; 3. miR-630降低细胞的自发凋亡及辐射诱导的细胞凋亡，miR-630亦可增强抗凋亡蛋白Bcl-2表达，同时抑制促凋亡蛋白Bax, Caspase 3, Caspase 7 and Caspase 9表达；4. miR-630可增强宫颈癌Hela细胞的致瘤性；5. miR-630可增强Hela细胞侵袭转移能力，同时通过降低上皮标志物E-cadherin和Cytokeratin的表达、升高间质标志物N-cadherin的表达来诱导上皮间质转化（EMT）的发生。.在完成本课题研究内容的同时，我们在课题实施中还发现乏氧也可以快速诱导Hela细胞中miR-630表达上调，并长时间维持miR-630的高表达水平。.目前课题取得的初步结论是：辐射和乏氧可诱导miR-630在宫颈癌Hela细胞中表达。miR-630可通过抑制辐照诱导的细胞凋亡增强宫颈癌Hela细胞的辐射抗性。miR-630可诱导宫颈癌Hela细胞发生EMT从而促进Hela细胞的侵袭转移。