非小细胞肺癌干细胞体外无血清培养模型的建立和TFEB调控非小细胞肺癌干细胞功能的机制研究

Lung cancer is notorious for its high occurrence and mortality.Previous reports show that there is a small group of indifferentiated cells so called cancer stem cells, which is pluripotent and considered to be the “cancer seeds”with no response to anti-cancer drugs. However, it is still elusive about the mechanisms of how these “cancer seeds”communicate with their microenvironment during the development of tumorigenesis. Therefore, we mimic the ischemic state during tumorigenesis by serum-free suspension culture. Through culturing lung cancer A549 cells in specific serum-free media, almost all the tumor spheres showed stemness’properties such as stem-cell morphology,expression of stem cell markers by 3D confocal images and enhanced tumorigencity in NOD-SCID nude mice injected with minimal amount of them. We also screened different signal molecules that significantly increased in those spheres, compared with common A549 cancer cells. Our data demonstrated that TFEB, a master regulator in cell autophage,was overexpressed. At the same time, the expression level of Bmi-1 was upregulated too. TFEB 3’UTR have two conserved sequences specifically for binding to miR-128. The occupation of miR-128 may reduce the inhibitory effect of miR-128 on Bmi-1, which promotes tumorigenesis since Bmi-1 plays a powerful role in the self-renewal and pluripotency of cancer stem cell (CSC).Taken together, we hypothesized that TFEB may act as a novel regulator of CSCs and would like to go deep our research of the underlying mechanisms. It will shed a light on TFEB function and its molecular role during the process of CSC self-renewal and pluripotency, which may serve as a novel drug target towards the eradication of lung CSCs.

肺癌干细胞被认为是导致肺癌患者复发、耐药和治疗失败的根源。已有文献报道认为，肿瘤异常增生过程中的缺血状态可促进肿瘤干细胞增多。但是，其中的分子机制尚不清楚。为此，申请人利用无血清悬浮培养法建立了肺腺癌A549细胞的干细胞模型。形态学检测、3D共聚焦扫描荧光标记的指示蛋白和裸鼠成瘤等实验都证明所得球状A549细胞具有肺癌干细胞的特征。通过筛选，申请人发现此A549干细胞中TFEB和Bmi-1表达显著增高。其中，由miRNA靶向预测, TFEB 3'-UTR可特异结合miR-128, 而减弱其对癌干细胞正调控因子Bmi-1 mRNA的降解。根据miRNA靶基因竞争学说，申请人推测TFEB mRNA通过竞争miR-128，从而上调Bmi-1和肺癌干细胞的表达。因此，本申请拟研究TFEB在分子细胞水平如何调控非小细胞肺癌干细胞的增生和自我更新。这将有助于了解非小细胞肺癌复发和耐药的根本原因。

肺癌干细胞被认为是存在于肺癌组织中的一小部分具有干细胞性质的细胞群体，具有自我更新和分化的能力，可形成不同分化程度的肺癌细胞，是肺癌生长、侵袭、转移和复发的根源，只有杀死肺癌干细胞才可以成功治愈肺癌。其中，非小细胞肺癌占据了肺癌的大部分。已有报道提示肿瘤异常增生过程中的缺血状态可促进肿瘤干细胞增多。但分子机制尚不清楚。为此，申请人利用无血清悬浮培养法建立了A549、H1299等多种非小细胞肺癌细胞的干细胞模型。形态学检测、3D共聚焦扫描荧光标记的指示蛋白和裸鼠成瘤等实验都证明所得球状非小细胞肺癌细胞具有肺癌干细胞的特征。通过筛选，申请人发现此类非小细胞肺癌干细胞中TFEB和Bmi-1表达显著增高，并通过生物信息学分析得出, TFEB 3'-UTR可特异结合miR-128, 而减弱其对癌干细胞正调控因子Bmi-1 mRNA的降解。因此，TFEB mRNA可通过竞争miR-128的靶基因，从而上调Bmi-1和肺癌干细胞的表达。本项目主要研究了TFEB调控非小细胞肺癌干细胞的增生和自我更新的作用，为解决非小细胞肺癌复发和耐药的难题提供了新思路。

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