长链非编码RNA NR_002331调控鼻咽癌细胞EMT参与铂类耐药的功能及机制研究

Chemoresistance is the main reason for treatment failure in nasopharyngeal carcinoma (NPC). In the present study, we screened for a new lncRNA NR_002331 which is significantly down-regulated in chemoresistant NPC cells and tissues through lncRNA microarray. NR_002331 could reverse the chemoresistance and Epithelial-Mesenchymal Transition (EMT) in the chemoresistant NPC cells. It could inhibit cell invasion and migration in vitro, too. Additionally, we found that EMT related molecular WNT2 was significantly down-regulated in NR_002331 over-expressed cells. Previous study showed that WNT2 proximal promoter is dominantly occupied by PRC2 component EZH2 and H3K27me3 histone modification, a marker for gene repression mediated by PRC2. Our result confirmed that NR_002331 could bind to EZH2 through RNA immunoprecipitation (RIP). Based on these findings, we further hypothesize that NR_002331 might down-regulate WNT2 through binding to EZH2, which lead to repressed PRC2-induced H3K27me3 histone modification of WNT2 proximal promoter. This study will futher investigate the function of NR_002331 through gene over-expression and down-regulation techonology and cell proliferation, migration and invasion assay. Moreover, RNA immunoprecipitation, detection of molecules in canonical Wnt signaling pathway, chromatin immunoprecipitation (ChIP)-quantitative real-time PCR and so on will be adopted to investigate the molecular mechanisms underlining NR_002331 function. Our study will provide new prespective in understanding NPC chemoresistance and will provide novel therapeutic targets for overcoming chemoresistance for NPC patients.

化疗耐药是鼻咽癌治疗失败的关键原因。课题组前期通过lncRNA芯片筛选出在鼻咽癌耐药细胞和组织中显著低表达的lncRNA NR_002331，在体外具有逆转细胞EMT和耐药、抑制细胞侵袭转移的功能；深入挖掘分子机制，EMT相关分子WNT2在NR_002331过表达细胞中显著下调，文献提示WNT2近端启动子主要由PRC2成员EZH2结合位点占领；RIP实验表明NR_002331可结合EZH2。由此提出如下科学问题：NR_002331是否通过EZH2介导WNT2启动子组蛋白H3K27me3修饰下调其表达，参与鼻咽癌细胞EMT和耐药过程？进一步拟采用基因过表达/沉默策略，通过体内外细胞增殖、转移等功能实验，结合RNA免疫共沉淀、染色质免疫共沉淀、经典Wnt通路分子表达等方法探寻分子机制。本项目有望揭示NR_002331调控EMT相关通路参与鼻咽癌顺铂耐药的新机制，可为克服鼻咽癌耐药提供新的思路。

非编码RNA鼻咽癌发生进展中发挥重要作用。课题组前期通过lncRNA芯片筛选出在鼻咽癌耐药细胞和组织中显著低表达的lncRNA NR_002331，在体外具有逆转细胞EMT和耐药、抑制细胞侵袭转移的功能；深入挖掘分子机制，EMT相关分子WNT2在NR_002331过表达细胞中显著下调，文献提示WNT2近端启动子主要由PRC2成员EZH2结合位点占领；RIP实验表明NR_002331可结合EZH2。进一步体内研究证实了NR_002331的生物学功能。为明确其作用机制，在鼻咽癌耐药细胞株中过表达NR_002331，该细胞株中回补WNT2可显著降低鼻咽癌细胞株对顺铂敏感性，且促进细胞侵袭、迁移。NR_002331调控WNT2的具体分子机制目前仍在进行中。此外，课题组依据前期筛选结果，进一步阐明了miR-203a-3p调控LASP1在鼻咽癌生长、增殖以及侵袭迁移中的具体作用和分子机制；通过检测LASP１蛋白水平表达及评估其与鼻咽癌患者预后参数关系，明确其在鼻咽癌进展过程中可能发挥的生物学功能。本项目有望为进一步提高鼻咽癌患者治疗疗效提供新的思路。