miR-19/GSK3β/β-catenin调控莱菔硫烷对肺癌干细胞干预作用的机制研究

Cancer stem cells (CSCs) play central role in the formation and development of cancer. To date, the involvement and function of miR-19, a key oncogenic miRNA, in regulating Wnt/β-catenin signal pathway and lung CSCs has not been defined. Meanwhile, the effects of sulforaphane on lung CSCs also remain to be explored. In our preliminary studies we found that lung CSCs upregulated miR-19a and miR-19b expression, decreased GSK3β expression level, and increased the expression levels of β-catenin as well as its target CSCs-related genes. We also showed that sulforaphane reduced the sphere formation of lung cancer cells cultured in serum-free-medium, decreased CSCs markers’ expression, decreased miR-19a and miR-19b expression, elevated GSK3β level, and suppressed β-catenin expression. However, the role and regulation of miR-19/GSK3β/β-catenin axis in lung CSCs activity and sulforaphane intervention is totally unknown. Using serum-free-medium cultured lung CSCs and nude mouse xenograft models, the proposed research will for the first time in the field to investigate the role of miR-19a/19b in GSK3β、β-catenin activation, target gene expression and lung CSCs activity. Regulation of Wnt/β-catenin signaling activity in lung CSCs activity will be examined. The transcriptional regulation of miR-19a/19b by p53 will be further explored. Moreover, this research will illustrate the role of miR-19/GSK3β/β-catenin in the interventional effects of sulforaphane on lung CSCs. Findings from this research will provide important new insights into the molecular mechanisms of miR-19/GSK3β/β-catenin axis in regulating lung CSCs activity as well as its target intervention.

肿瘤干细胞对肿瘤发生、发展具决定性作用。有关miR-19调控Wnt/β-catenin通路和肺癌干细胞、以及莱菔硫烷对肺癌干细胞的干预作用迄今未见任何研究报道。我们前期研究发现，肺癌干细胞活性与其上调miR-19a/19b、抑制GSK3β、激活β-catenin并影响其靶基因表达有关；莱菔硫烷可抑制肺癌干细胞活性；然miR-19/GSK3β/β-catenin是否真正介导肺癌干细胞活性及莱菔硫烷的干预效应尚属未知。本课题拟采用体外细胞模型和动物模型相结合的方法，在领域内创新性地探讨肺癌干细胞miR-19对GSK3β、β-catenin活性、肺癌干细胞靶基因表达的影响、作用及调控机制，并在此基础上研究miR-19/GSK3β/β-catenin在莱菔硫烷干预肺癌干细胞效应中的调控作用。旨在为探索肺癌干细胞的调控机制，进而通过靶向作用于肺癌干细胞的关键分子而干预肺癌提供新的研究策略和科学依据。

肿瘤干细胞在肿瘤发生、发展过程中具有决定性意义。Wnt/β-catenin通路是调控肿瘤干细胞的关键信号通路。miRNAs对基因表达及其功能具有重要的调控作用。莱菔硫烷是十字花科植物中具有抗癌作用的主要活性成分。迄今有关致癌性miR-19调控Wnt/β-catenin通路和肺癌干细胞活性、以及莱菔硫烷对肺癌干细胞的干预作用未见任何研究报道。本课题采用肺癌细胞无血清悬浮培养克隆成球的体外模型及裸鼠移植瘤模型相结合的方法，深入探讨了miR-19对Wnt/β-catenin通路、肺癌干细胞活性、靶基因表达的影响、作用及其调控机制，并在此基础上研究miR-19/GSK3β/β-catenin在莱菔硫烷干预肺癌干细胞效应中的调控作用。本研究发现miR-19a/miR-19b在肺癌干细胞中高表达；过表达miR-19a/miR-19b显著促进肺癌干细胞的活性，而低表达miR-19a/miR-19b则明显抑制肺癌干细胞活性，表明miR-19a/miR-19b对肺癌干细胞的正向调控作用。肺癌干细胞中Wnt/β-catenin通路对维持其干性具有重要作用。本研究首次提出Wnt/β-catenin通路的关键负调控因子GSK3β是miR-19a/miR-19b的靶基因，miR-19a/miR-19b通过直接靶向抑制GSK3β进而激活该通路。细胞实验结果显示，莱菔硫烷具有显著抑制肺癌干细胞活性的作用，miR-19/GSK3β/β-catenin通路介导莱菔硫烷对肺癌干细胞的干预作用。裸鼠移植瘤实验结果亦表明了莱菔硫烷对肺癌干细胞及miR-19/GSK3β/β-catenin轴的抑制作用。综上所述，本项目研究为阐明肺癌发生发展过程起关键作用的肺癌干细胞的调控机制和其关键靶点，进而为以机制为导向的干预研究提供了新的重要科学依据和策略。