沉默ΔNp63α表达诱导食管鳞癌干细胞分化的作用及相关机制研究

The precise roles of p63 in tumour suppression have been hotly debated. Although some studies show p63 overexpression in human cancer，some demonstrate a loss of p63 associated with tumour progression and metastasis. Much of this controversy is due to the existence of multiple isoforms. The full-length TA isoform of p63 bears structural and functional similarity to p53, whereas the ΔN isoforms of p63, which lack the transactivation (TA) domain, act primarily in dominant-negative fashion against p53, TAp63 and TAp73.These activities suggest that TAp63 is a tumour suppressor gene and ΔNp63 is an oncogene.There is a strong relationship between the high expression of ΔNp63α ,a isoforms of p63 ,and the generation and development of esophageal squamous cell carcinoma.In our previous research,we found that as a controlling gene of esophageal squamous cell carcinoma, ΔNp63α can regulate some feature belonging to the aspect of the esophageal squamous cell carcinoma stem cells (ESCSCs) as a kind of stem cell. There is no literature report whether we can inhibit the growth and migration of the esophageal squamous cell carcinoma by silencing the expression of ΔNp63α in order to induce the ESCSCs to differentiate to the terminal cell.This research apply a series of methods of cytobiology and molecular biology to study 1) the influence that scilening the expression of ΔNp63α made to the ESCSCs's self-renewal,multi-directional differentiation and sensitivity of chemotherapy; 2)The mechanism of signal transduction in the process that silencing the expression of ΔNp63α induce the differentiation of ESCSCs and, consequently, promote the apoptosis of the tumor cell;3)the inhibition of the growth and migration of the tumor which is caused by silencing the expression of ΔNp63α.Based on the new viewpoint that we can treat the ESCSCs with the methods provided by differentiation therapy,our research aim to reveal the possible mechanism and the effect of silencing the expression of ΔNp63α in the targeted therapy of the ESCSCs.

复层上皮样干细胞标记物p63基因的ΔNp63α亚型高表达与食管鳞癌发生、发展密切相关。我们前期研究发现，ΔNp63α能够调控食管鳞癌干细胞（ESCSCs）的某些干细胞特性，是食管鳞癌"干性"调控基因。但沉默ΔNp63α的表达，是否可以诱导ESCSCs向终末细胞分化，从而抑制食管鳞癌的生长和转移，未见相关文献报道。本研究通过系列细胞生物学及分子生物学实验方法，①研究沉默ΔNp63α表达对ESCSCs自我更新、多向分化、化疗敏感性等生物学特性的影响；②研究沉默ΔNp63α基因诱导ESCSCs分化从而促进肿瘤细胞凋亡的信号传导机制；③在体外ESCSCs小鼠移植瘤模型中，深入研究沉默ΔNp63α表达对肿瘤干细胞特性的影响及对肿瘤生长、转移的抑制作用。本研究立足于对ESCSCs进行分化治疗这个新视点，旨在揭示沉默"干性"基因ΔNp63α表达在ESCSCs靶向治疗中的效果和可能机制。

本项目首先通过免疫组织化学SABC法检测304例食管鳞癌组织中ΔNp63α表达水平及与临床病理的相关性，探讨原癌基因ΔNp63α在食管鳞癌中的表达与临床预后的相关性。通过随访，进行生存分析，我们发现ΔNp63α高表达的患者生存期限显著低于其他食管鳞癌患者；进而通过多变量Cox比例风险回归模型最终确定ΔNp63α是影响食管鳞癌患者预后的独立危险因素。.本研究以神经营养素低亲和力受体p75NTR（Neurotrophin receptor p75，p75NTR）作为食管鳞癌干细胞标志蛋白分离食管鳞癌干细胞；进一步通过细胞学及动物学实验研究，提示沉默ΔNp63α基因的表达，对食管鳞癌干细胞具有诱导分化作用，肿瘤干细胞数量减少，终末细胞分化增多。明确了“干性”调控基因ΔNp63α对食管鳞癌细胞增殖、侵袭及转移致瘤能力的影响。.鉴于肿瘤干细胞中普遍存在相关信号通路的活化，并且活化相应通道与细胞自我更新和多向分化密切相关。本研究采用Western印迹法检测RNA干扰抑制ΔNp63α蛋白表达后对Wnt、Notch、Sonic hedgehog (Sh)信号传导通路中关键蛋白β-catenin、Notch1、Smo蛋白表达的影响。结果显示，抑制食管鳞癌细胞中ΔNp63α 蛋白表达后后，β-catenin、Notch1、Smo表达显著下降，提示ΔNp63α可能通过Wnt、Notch、Sonic hedgehog (Sh)信号传导通路调控食管鳞癌细胞的生物学活性。鉴于β-catenin表达下调幅度较大，我们后续对ΔNp63α经由Wnt/β-catenin信号通路的影响食管鳞癌细胞的作用开展了进一步研究。在研究中，我们首先通过抑制食管鳞癌干细胞中ΔNp63α的表达，研究Wnt/β-catenin变化情况，随后抑制β-catenin的表达，观察食管鳞癌干细胞中ΔNp63α的表达情况。研究结果从体外研究及体内研究两个方面初步证明ΔNp63α基因对β-catenin具有上游调控作用，即ΔNp63α作为“干性”调控基因通过对β-catenin的调控，维持肿瘤干细胞的“干性”功能；但其具体机制有待进一步研究。.目前本项目已完成SCI收录论著4篇，IF：10分，单篇最高IF：3.840分，中文核心期刊收录论著4篇，全国大会发言1次。本研究已申请国家发明专利2项。