miR-324-3p/WNT2B介导上皮-间质转化参与鼻咽癌放疗抗拒的功能和分子机制研究

Radioresistance severely restricts the clinical management of nasopharyngeal carcinoma (NPC). Our previous investigation have demonstrated that NPC cells underwent EMT during the exposure of irradiation. Overexpression of miR-324-3p is observed in radioresistant NPC cells compared to their parental cells. In vitro functional analysis reveals that miR-324-3p regulates the radioresistance of NPC and participates in the process of EMT. The expression of WNT2B is also directly regulated by miR-324-3p. Therefore, we hypothesize that miR-324-3p regulates the radioresistance of NPC via WNT2B mediated-EMT. For this purpose, NPC radioresistance cells with stable miR-324-3p overexpression and their parental cells with stable miR-324-3p inhibition will be established, and the impacts of miR-324-3p on NPC radioresistance, EMT and WNT2B signaling pathway will be observed. Then EMT is to be induced in NPC cells, or the WNT2B signaling pathway is to be restored in miR-324-3p overexpressing radioresistant NPC cells, the alterations of radioresistance will be investigated. Finally, the clinical significance of miR-324-3p and proteins of WNT2B signaling pathway is evaluated. The propasal aims to discover the molecular biomarkers and therapeutic targets associated with NPC radioresistance.

鼻咽癌放疗抗拒为目前临床与基础研究中的热点和难点。课题组发现鼻咽癌细胞在照射过程中发生上皮-间质转化(EMT)，并从所得的放疗抗拒miRNA差异表达谱中发现miR-324-3p能在体外调控鼻咽癌的放疗抗拒及EMT改变，对WNT2B具有靶向调控作用。结合现有研究进展，我们假设miR-324-3p可通过WNT2B信号通路调控鼻咽癌EMT进而影响其放疗抗拒性。为此，本项目拟构建miR-324-3p稳定表达及沉默的鼻咽癌细胞，探讨miR-324-3p对鼻咽癌放疗抗拒、EMT及WNT2B通路的影响，并基因芯片分析miR-324-3p改变所致的下游差异表达基因；进一步诱导EMT改变、恢复WNT2B表达及下游信号通路活化确定其对miR-324-3p介导的鼻咽癌放疗抗拒性改变的影响；收集大样本临床标本验证miR-324-3p及WNT2B的临床价值，力求找到与鼻咽癌放疗抗拒性密切相关的干预靶点和分子标志物。

鼻咽癌放疗抗拒为目前临床与基础研究中的热点和难点。课题组发现鼻咽癌细胞在照射过程中发生上皮-间质转化(EMT)，并从所得的放疗抗拒miRNA差异表达谱中发现miR-324-3p能在体外调控鼻咽癌的放疗抗拒及EMT改变，对WNT2B具有靶向调控作用。结合现有研究进展，我们假设miR-324-3p可通过WNT2B信号通路调控鼻咽癌EMT进而影响其放疗抗拒性。为此，本项目拟构建miR-324-3p稳定表达及沉默的鼻咽癌细胞，探讨miR-324-3p对鼻咽癌放疗抗拒、EMT及WNT2B通路的影响，并基因芯片分析miR-324-3p改变所致的下游差异表达基因；进一步诱导EMT改变、恢复WNT2B表达及下游信号通路活化确定其对miR-324-3p介导的鼻咽癌放疗抗拒性改变的影响；收集大样本临床标本验证miR-324-3p及WNT2B的临床价值，力求找到与鼻咽癌放疗抗拒性密切相关的干预靶点和分子标志物。通过相关实验，课题组发现以下成果：（1）miRNA-324-3p与WNT2B的表达水平提示鼻咽癌的恶性程度；（2）miRNA-324-3p通过WNT信号通路介导鼻咽癌的EMT和侵袭转移；（3）WNT2B亦受到miRNA-185-3p的表达水平影响，介导鼻咽癌的放疗抵抗性、EMT和侵袭转移；（4）miRNA-324-3p介导放疗抵抗其他机制的探索，miRNA-324-3p表达水平不同细胞株相关lncRNA表达谱的构建。尽管获得的基金资助比预期有所减少，但根据相关实验数据进行了资料整理，数据统计，共发表论文15篇，其中SCI 12篇，专利5个，获得省部级奖项1项，厅级奖励1项。共培养博士3人，硕士5人。完成了拟定的大部分目标，并结合了新进展展开了lncRNA相关的前沿领域研究，为后续鼻咽癌放疗抵抗机制的研究提供了更为丰富的理论和经验。