结直肠癌中MicroRNA-10b/Beclin 1/Parkin信号通路调控线粒体自噬的机制研究

Although autophagy is one highly conserved phenomena in evolution, its role in cancer has been controversial. Our previous study indicated that both dysfunctional mitochondrial frequency and expression of key autophagic protein had changed significantly in colorectal cancer; we therefore proposed one “autophgic signature” model, further analysis suggested this autophagic signature strongly correlated with overall survivals of cancer patients. Here we focused on the role of Beclin 1, one key protein in autophgic process, during carcinogenics. MicroRNA-10b was identified as the upstream regulator of Beclin 1 by Lenti-miRNA lentiviral library screening, while co-immunoprecipitation results revealed that Parkin could bind to Beclin 1 directly. Based on these results and other reported studies, we believe that microRNA-10b/Beclin 1/Parkin pathway regulate mitophagy in cancer cell, thus affect the development of colorectal tumor. This project will validate this hypothesis through several levels of investigations including cellular, animal and clinical experiments, so the mechanism of how mitophagic disorders could be clarified in the cancer cells. The successful completion of this project will provide theoretical foundation for new target of anti-tumor drugs; on the other hand, by evaluating the role of key molecules in in cancer clinical staging, treatment and prognosis, etc. sensitivity as bio-markers, the current study could provide clinical evidence for future personalized medicine.

自噬是一种进化上高度保守的生命现象，但其在肿瘤中的作用存在争议。本课题组前期工作表明，结直肠癌中受损线粒体的数量及自噬关键蛋白的表达都出现异常；并由此提出“自噬标识”这一概念，分析表明自噬标识与患者生存期具有显著相关性。本项目以重要自噬蛋白Beclin 1为切入点，通过Lenti-miRNA慢病毒库筛选出其上游结合分子microRNA-10b，同时发现Beclin 1与Parkin可直接结合；我们猜想microRNA-10b/Beclin 1/Parkin通路具有调控线粒体自噬的功能，从而影响肿瘤的发生发展。本课题将通过细胞、动物及临床等多层次的研究验证上述理论，阐明线粒体自噬紊乱的产生机制。本项目的完成不但会为抗癌药物的研发提供新的特异性靶点并奠定相应的理论基础；同时通过评价关键分子在临床分期、治疗敏感性以及预后预测等方面作为生物标记物的潜在价值，为个体化医疗的实现提供临床依据。

本项目系统的阐述了细胞自噬过程中重要基因Beclin 1、LC 3以及Bcl-xL在结直肠癌发生发展过程中的调控作用机制；同时利用课题组丰富的临床样品资源，明确了这些基因在结直肠癌预后转移、复发以及生存期中作为生物标记物的重要价值。课题组确定了临床结直肠癌样品中中受损线粒体与生存期具有明显相关性；并研究了肿瘤组织中心部分与癌旁组织中Beclin 1、LC 3以及Bcl-xL的表达与患者生存期之间的密切关系，并以这些自噬相关分子为基础开创性的提出了“自噬标识”这一概念，并计算患者五年期生存期的风险度。结合传统临床使用的TNM分期法则，衡量两者独立或者相互叠加在生存期预测方面的敏感性和特异性。检测miRNA-10b的表达与临床分期以及生存期之间的相关性；阐明了miRNA-10b对Beclin 1的表达以及结直肠癌细胞侵袭特性的影响。验证缺氧条件下，miRNA-10b抑制剂可在肿瘤细胞系中上调自噬活动，而沉默Beclin1后则可部分恢复由miRNA-10b抑制剂产生的自噬活动。该研究结果不但在结直肠癌的临床实践中具有一定的指导意义，同时也预示着自噬在肿瘤的后期发展过程中起到了重要作用；而且我们还推测从结直肠癌中得到的这些结论可能也会适用于其他类型的肿瘤。本项目的完成不但为抗癌药物的研发提供新的特异性靶点并奠定相应的理论基础；同时通过评价关键分子在临床分期、治疗敏感性以及预后预测等方面作为生物标记物的潜在价值，为个体化医疗的实现提供了临床依据。本课题项目已按计划要求完成考核指标，在肿瘤专业国际学术刊物上发表SCI收录论文8篇。