甲状腺癌BRAF V600E在DRP1介导的线粒体稳态中的调控新机制研究

The braf gene had a high mutation rate in papillary thyroid cancer (PTC) and the mutation activated its kinase activity and caused the constant activation of MAPK pathway, so resulted in abnormal tumor proliferation. However, clinical research showed that BRAF kinase inhibitors could not completely alleviate the condition of thyroid cancer patients, revealing BRAF probably has other kinase-independent activity. Experiment results showed that both BRAF WT and BRAF V600E located on mitochondria, but only BRAF V600E promoted the expression of DRP1 and BRAF kinase inhibitors were unable to block the autophagy enhancing effects of BRAF V600E. So we propose the hypothesis that the kinase-independent activity of BRAF V600E regulates the mitochondrial homeostasis mediated by DRP1, so as to reveal the new function of BRAF V600E in the development of thyroid cancer. Firstly, we intend to determine the mitochondrial location of BRAF and its effects on morphology and function of mitochondria. Secondly, the relation between BRAF mutation and the expression and phosphorylation of DRP1 will be investigated in PTC samples. Finally, by advanced molecular biological methods, such as overexpression, knockdown and co-IP, the mechanism which BRAF V600E promotes mitochondrial fission and enhances autophagy to maintain mitochondria homeostasis through kinase-independent activity will be investigated. This study provides a potential molecular target for therapy of thyroid cancer.

甲状腺乳头状癌（PTC）中braf基因高突变，使得其激酶活性和MAPK通路被激活，造成肿瘤异常增殖。临床研究显示BRAF激酶抑制剂不能完全缓解PTC病人的病情，揭示BRAF尚存在激酶非依赖活性。预实验证明BRAF WT和BRAF V600E都定位于线粒体，但只有BRAF V600E促进DRP1的高表达，且BRAF抑制剂不能阻止BRAF V600E对自噬的增强。因此，我们提出“甲状腺癌中BRAF V600E通过激酶非依赖活性调控DRP1介导的线粒体稳态”的科学假说，以期揭示BRAF在甲状腺癌发生发展中的新功能。本项目拟确定BRAF的线粒体定位及对线粒体形态和功能的影响；分析临床PTC样本BRAF突变与DRP1的表达及磷酸化的相关性；利用过表达、敲除、免疫共沉淀等手段探索BRAF V600E通过其激酶非依赖活性促进线粒体分裂，增强自噬，维持线粒体稳态的分子机理，为甲状腺癌的治疗提供新的靶标。

甲状腺乳头状癌（PTC）中braf基因高突变, 使得其激酶活性和MAPK通路被激活，造成肿瘤异常增殖。我们的研究揭示了BRAF V600E突变和甲状腺癌代谢重编程之间的关系。在本课题中我们通过构建BRAF V600E过表达和BRAF V600E敲降的甲状腺细胞系，来研究其对线粒体呼吸和糖酵解等功能的影响。我们的研究结果表明，过表达BRAF V600E抑制OXPHOS基因表达和线粒体呼吸，增强糖酵解水平。BRAF V600E突变的临床甲状腺癌样本中PGC-1β的表达降低，但是HIF1α的表达增强。MEK1的抑制剂处理增强了BRAF V600E突变的甲状腺癌的线粒体呼吸。另外，HIF1α是BRAF V600E的一个下游靶点，通过MYC调控PGC-1β的表达。而且，糖酵解相关的酶，例如LDHA和PKM2，在BRAF V600E突变的甲状腺癌样本中表达上调，糖酵解水平增加。本研究的研究结果表明，甲状腺癌中BRAF V600E突变通过HIF1α-MYC-PGC-1β降低线粒体呼吸，增强糖酵解。总之，本课题阐述了BRAF V600E和甲状腺癌代谢重编程之间的关系，研究了BRAF V600E调控甲状腺癌代谢重塑的分子机制，解析了BRAF V600E调控线粒体代谢的新机制，为甲状腺癌研究奠定理论基础和提供可能的干预靶点。受该项目资助共发表5篇研究论文，分别发表在FEBS Journal, Biomedicine & Pharmacotherapy, Food and Function, Journal of cell science杂志上。依据该项目内容撰写一项发明专利，专利正在申请中，共培育2名硕士研究生。

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