NF-κB/CXCR4通路激活自噬维持鼻咽癌干细胞干性及EGCG对其干预的研究

Cancer stem cells (CSCs)is the basic cause of nasopharyngeal carcinoma(NPC) invasion, metastasis and recurrence, autophagy is one of the important survival mechanism of cellular responsing to the stress environmental. Our previous studies have found that there is a higher autophagy in nasopharyngeal carcinoma stem cells(NPCSC), also found that NF-κB/CXCR4 signaling pathway plays an important role in maintaining stemness of NPCSC and some studies have shown that CXCR4 can activate an important initiation factor, PI3KⅢ, activating autophagy through the downstream signaling, suggesting that NF-κB/CXCR4 is closely related with the autophagy and plays a critical role in the regulation of NPCSC function .Natural tea polyphenols EGCG has the Characteristics of low toxicity and high efficiency and shows good potential in the treatment of many diseases through the regulation of autophagy ,but that EGCG inhibits NPCSC through the regulation of autophagy is unclear. Based on this, this research is proposed to study the relation of cell autophagy and NF-κB/CXCR4 pathway and its role in maintaining NPCSC stemness as the breakthrough point, we will further combine natural plant ingredients, CSCs, autophagy activation mechanism to explore. This research has expanding and innovative theoretical value , research results help to clarify the development mechanism of NPC and to provide theoretical basis on researching and developing innovation, low toxicity and efficient new drug targeting to CSCs specific targets .

肿瘤干细胞是鼻咽癌侵袭、转移、复发的根源,自噬是细胞应对应力环境因素的重要生存机制之一。我们前期研究发现鼻咽癌干细胞存在较高水平自噬，同时发现NF-κB/CXCR4信号通路在维持鼻咽癌干细胞干性中发挥着重要作用，而研究显示CXCR4能激活下游信号的重要启动因子PI3KⅢ激活自噬，推测NF-κB/CXCR4与自噬关系密切并在鼻咽癌干细胞功能调控中起着关键作用。天然茶多酚EGCG低毒、高效，通过调控自噬在治疗诸多疾病中展现良好潜能，但EGCG抑制鼻咽癌干细胞是否与自噬有关尚不明确。基于此，本课题拟研究细胞自噬与NF-κB/CXCR4通路关系及其在鼻咽癌干细胞干性维持中的作用，并以此为切入点，进一步将天然植物成分、肿瘤干细胞、自噬激活机制三者有机结合起来探索。课题理论上具有拓展性及创新性，研究结果有助于阐明鼻咽癌的发生发展机制，为创新、低毒、高效的靶向肿瘤干细胞特异靶点的新药研发提供理论依据。

肿瘤干细胞是鼻咽癌侵袭、转移、复发的根源,自噬是细胞应对应力环境因素的重要生存机制。课题采用无血清悬浮培养法富集鼻咽癌干细胞，通过体内外实验对鼻咽癌干细胞进行干性鉴定并检测分析其自噬水平，再采用RNAi干扰、通路阻断剂等实验技术研究自噬、信号通路与肿瘤干细胞干性的作用及机制，并初步探索了天然茶多酚EGCG抑制鼻咽癌干细胞干性的作用及机制。结果提示：无血清悬浮培养法成功获得鼻咽癌干细胞，其具有较强的自我更新、侵袭、转移、成瘤等干细胞干性；与亲本细胞比较，鼻咽癌干细胞中NF-κB/p65蛋白的表达明显增强，沉默NF-κB/p65的表达，鼻咽癌干细胞的克隆形成率、迁移侵袭力、体内成瘤率均明显减低(P均＜0.01)；鼻咽癌CNE2干细胞自噬小体形成明显增多(P<0.01),并高表达自噬相关基因(Beclin1、LC3B)(P<0.01)，调控Beclin1影响自噬继而调控鼻咽癌干细胞克隆形成、侵袭、迁移能力，结果提示鼻咽癌干细胞较其亲本细胞具有较高自噬水平，自噬影响肿瘤干细胞干性；研究还初步揭示EGCG通过影响NF-κB/p65活性及其对Twist1的转录表达发挥抑制鼻咽癌干细胞干性的功能及机制。在以上研究基础上，进行了本课题相关延伸研究：成功富集获得肺腺癌A549干细胞，其同样具有肿瘤干细胞干性及较高自噬水平，并发现长链非编码RNALUCAT1(LncRNALUCAT1)在肺腺癌A549干细胞中明显高表达，其正向调控Beclin-1基因的转录表达激活自噬，进而维持肺腺癌干细胞干性；与亲本细胞比较，CNE-2干细胞中HIF-1α和NF-κB/p65蛋白表达明显增高(P＜0.01)，X射线辐射干细胞后各放射参数D0、Dq、N及SF2均升高(P＜0.01)；2-甲氧基雌二醇(2-MeOE2)干预CNE-2干细胞后，干细胞增殖、迁移能力呈浓度依赖性降低(P＜0.01)，放射增敏比增高。综上所述，课题研究结果为今后深入、广泛开展肿瘤干细胞相关研究打下实验及理论基础，为创新、低毒、高效的靶向肿瘤干细胞特异靶点的新药研发提供了理论依据。