miR-146a/LIN28/Let-7反馈轴调节肺癌干细胞自我更新能力的作用及机制研究

The incidence of Lung cancer is much higher than that of other malignancies, and lung cancer always results in poorer prognosis. Decreasing the ratio of lung cancer stem cells (LCSCs) is of great value to achieve clinical cure of patients with lung cancer. In previous studies, we found that miR-146a decreased the ratio of LCSCs, with increased Let-7 level, of which we have identified the suppressive roles exhibiting on Wnt signaling activation; however, the roles of miR-146a in the procss of self-renewal of LCSCs, and the relationship with Wnt signaling, which has been tentatively applied in preclinical trials, is still unclear. We predicted the potential binding site between the 3’UTR of LIN28A and miR-146a, and also found that Wnt activator may funcion as Let-7 sponges in LCSCs, with the results that Let-7 retained the same level, when its downstream genes increased significantly. Based on these results, we hypothesized that miR-146a suppresses the self-renewal of lung caner stem cells through targeting and degrading LIN28A, which help to increase Let-7 consequently, with Wnt signaling inactivation. Besides, the overexpressed miR-146a reversed the sponges regulation of Wnt activator on Let-7. To prove this hypothesis in this project, we plan to explore the functions and mechanisms of miR-146a in Let-7 induction of Wnt signaling blockade, by using stem cells isolation and identification, luc-assay, western blot and fluorescent probe technique real time PCR. The results of this study will help to target and block the Wnt signaling sustaining of self-renewal of cancer stem cells via non-coding RNAs, and pave the way to the consequent clinical cure of lung cancer with basis of new thoughts, new methods and theoretical foundation.

肺癌发病率高预后差，降低肺癌干细胞比例对实现肺癌临床治愈具有重要意义。前期我们发现miR-146a可降低肺癌干细胞比例，其过表达伴随Wnt通路抑制因子Let-7的升高，而miR-146a在肺癌干细胞自我更新过程中的作用及其与Wnt通路的关系未见报道。前期生物信息学预测miR-146a可与LIN28A非编码区结合；肺癌细胞中Wnt通路激活对Let-7起海绵调性节作用，因此我们提出假设，miR-146a通过靶向抑制LIN28表达而上调干细胞内成熟Let-7表达，减弱Wnt通路活性及其维持的肺癌干细胞自我更新；miR-146a逆转肺癌干细胞中Wnt激活剂对Let-7的海绵性调节作用。课题拟通过荧光素酶报告实验、荧光探针等方法阐明miR-146a通过上调Let-7而阻断干细胞中Wnt通路的激活，为进一步以非编码RNA为手段而靶向Wnt通路，抑制肺癌干细胞的自我更新能力提供新思路、新方法和理论依据。

肺癌组织对化疗的反应决定长期治疗效果，无论患者有无手术计会，如何减少肺癌患者临床耐药的发生，降低肺癌远期复发率或维持疾病缓解率，延长患者的生存期，成为对抗肺癌亟待解决的棘手问题。肺癌干细胞对肺癌细胞群体的长期生存、耐药及复发起重要作用，被誉为肿瘤的种子，因而降低肺癌干细胞的比例及数量，在杀伤肺癌细胞的同时，抑制肺癌干细胞造成的肺癌细胞群体再生，对于增敏抗癌治疗效果，根治肺癌具有重要的意义和应用价值。课题主要包括三部分内容，分别关于基础实验，动物体外实验，以及临床样本内相关指标的检测分析，首先围绕miR-146功能与机制的相关研究，以miR-146作为承前启后的重要节点分子，结合Let-7在肺癌干细胞中的功能研究，探索Wnt通路激活依赖特定非编码RNA对 Let-7 功能的竞争性抑制，形成自身正反馈调节环路，促进肺癌干细胞的对称分裂、复制增殖以及体内成瘤，而miR-146a 过表达通过阻断 Wnt 通路激活而减弱干细胞的自我更新能力。机制探索分为两部分内容，分别在肺癌及乳腺癌细胞中，探究相关目的基因在干细胞亚群中的作用，首先我们探索了可能对Let-7有海绵吸附及调节作用的ceRNAs，探索发现CCAT1通过Let-7的竞争性抑制，在肺癌中促进Wnt通路调控的干细胞亚群自我更新，促进了干细胞亚群的对称分裂，且Wnt通路激活后对CCAT1有明显的正向调控作用，而后课题组验证了H19在激素依赖的乳腺癌干细胞自我更新中的关键作用仍旧依赖于对Let-7的竞争性抑制，证实Wnt通路在Let-7下游功能的发挥中受到上游ceRNAs的调控支配。通过多种细胞系的验证，课题的临床分析及基础实验均已完成，下一步体内实验及更为深入的研究计划也有序开展，探索多种对肿瘤干细胞亚群有调控作用的因素，利用肿瘤细胞不同周期的增殖特性，可有效地抑制其增殖，阻断分裂更新，有助于改善治疗的敏感性，甚至治愈肿瘤。本研究提示了非编码RNA的重要作用，而不同类型的非编码RNA之间彼此相互影响，形成了复杂的调控环路，精细的调控肿瘤干细胞发生、维持及演变的多个环节。