白藜芦醇通过miR-330靶向调控MRP1诱导鼻咽癌放射增敏的研究

Nasopharyngeal Carcinoma (NPC) caused prevalent deaths in south china and its incidence rate continues to rise. Ionizing radiation (IR) has been a widely used approach to NPC therapy; responses of NPC to radiotherapy depend on the radiosensitivity of the tumor cells. Resveratrol (Res), Chinese herb monomer extracts, has an effect on radiosensitization; however, its molecular mechanism has yet to be clear. Our previous data showed that Res treatment caused growth inhibition and apoptosis induction of NPC; notably, Res promoted NPC cell sensitivity to IR via downregulation of MRP1. Then, what molecular signaling pathways get involved in the downregulation of MRP1 followed by Res treatment inducing radiosensitization? Bioinformatics software predicted MRP1 to be miR-330-targeting gene. Furthermore, Luciferase reporter gene assay and cellular transfection experiment confirmed that miR-330 directly targeted MRP1 for its downregulation. Moreover, Real Time Q-PCR results indicated the upregulation of miR-330 in NPC cells treated with Res. Therefore, we speculated that pretreatment of Res sensitized NPC cells to radiation through downregulation MRP1 targeted by miR-330. This proposal will employ cellular experiments, tumor animal models and NPC clinical tissues to illustrate further the molecular mechanism by which Res enhanced radiosensitivity of NPC, and provide the scientific ground for Chinese medicine adjuvant therapy and novel miRNA-targeted treatment of tumor..

鼻咽癌是中国南方常见恶性肿瘤并且其发生率持续升高，放射疗法为其治疗的主要手段，然其疗效仍依赖于肿瘤细胞放射敏感性。中药单体白藜芦醇具有放射增敏作用，但其分子机制尚待明确。我们前期研究表明白藜芦醇可以抑制鼻咽癌细胞生长，诱导细胞凋亡，并能通过下调MRP1表达而实现放射增敏。那么，白藜芦醇如何下调MRP1诱导放射增敏？软件预测提示MRP1是miR-330靶基因，进一步，荧光报告基因及细胞内实验均证实miR-330直接靶向调控MRP1。另外，定量PCR结果表明白藜芦醇能够诱导鼻咽癌细胞miR-330显著上调。由此，我们首次提出白藜芦醇通过miR-330靶向调控MRP1，进而诱导鼻咽癌放射增敏的可能性。本项目拟采用细胞体外实验、肿瘤动物模型以及人体鼻咽癌病理组织获得白藜芦醇诱导鼻咽癌放射增敏及其分子机制的可靠证据，为应用中药辅助肿瘤治疗及miRNA作为全新的肿瘤治疗靶点提供有力的科学依据。

鼻咽癌在中国华南地区发病率较高，目前治疗手段主要是放疗或放化疗联合。放疗抵抗是个瓶颈制约放疗效果。天然多酚化合物-白藜芦醇具有抗氧化，保护心血管等药效，在保健品市场中受追捧。虽然近来很多研究提出藜芦醇具有抗肿瘤效果，但是剂量普遍较高，具有细胞毒性。本研究我们优化白藜芦醇药物浓度（50 μM），确保对鼻咽癌细胞活力没有影响，另外还可以将CNE-1细胞周期阻滞到G1期，诱导鼻炎癌细胞放疗增敏。为了回答究竟白藜芦醇是否能够放疗增敏，我们设计了体内外实验。体外，和单独照射相比，白藜芦醇联合放射显著抑制CNE-1克隆形成并促进细胞凋亡；体内，白藜芦醇和放疗联合组肿瘤体积和肿瘤均比单独照射组显著减小，而裸鼠的体重没有明显变化，进一步说明白藜芦醇毒性较低。分子机制方面，我们已经筛选到白藜芦醇和放疗能够协同上调miRNA-330上调，其下游靶基因是E2F1。免疫印迹显示：白藜芦醇处理CNE-1细胞，E2F1显著下调。为了回答E2F1是否介导白藜芦醇放疗增敏，我们用慢病毒方法建立过表达和干扰E2F1鼻咽癌细胞株。干扰E2F1表达，CNE-1细胞呈现放疗增敏；过表达E2F1后能够逆转白藜芦醇诱导放疗增敏。结果说明白藜芦醇通过下调E2F1诱导放疗增敏。白藜芦醇低毒安全，来源丰富。我们优化其剂量，体内外实验均取得了较好的放疗增敏效果。这为白藜芦醇应用于放疗提供理论支持。