p53差异转录调控GPC1/miR-149-5p介导胃癌耐药新机制的研究

Chemotherapy resistance is the important reason for gastric cancer(GC) related death. We previously found that miR-149-5p targets CXCR1, and CXCR1 promotes ERK and AKT phosphorylation and mediates chemotherapy resistance in GC. With the help of proteomics and mass spectrometry experiments, we identified some CXCR1 silencing related new target proteins (hnRNPK, HSP27 and sorcin),which directly or indirectly mediated cell cycle, mitochondrial and FASL /FAS apoptosis pathway. Bioinformatics analysis showed that there is one potential p53 binding domain in the promoter region of host gene GPC1 of miR-149-5p; and mutant type p53 regulates chemotherapy resistance in tumors. Therefore, on the above basis, in this project we put forward the following scientific hypotheses: the wild type and mutant type p53 differently regulated transcription of GPC1 and miR-149-5p, and p53/GPC1/miR-149-5p-CXCR1 regulating axes, on the one hand, mediates mitochondria and FASL/FAS apoptosis pathway crosstalk by CXCR1 promoting ERK and FAK/AKT pathway, and then prompting hnRNPK, HSP27 and sorcin expression, which causes the cells to evade apoptosis induced by chemotherapy; On the other hand, GPC1 and CXCR1 promote cell cycle through crosstalk, eventually leading to chemotherapy resistance in GC. We intend to verify the above scientific hypothesis from four levels of cells - animal models - molecules - clinical samples. The project will elucidate the new molecular mechanism of p53 mediating chemotherapy resistance through GPC1/miR-149-5p.

耐药是胃癌致死的重要原因。我们前期发现miR-149-5p靶向CXCR1；CXCR1促ERK和AKT磷酸化，介导胃癌耐药。前期蛋白质组学鉴定出CXCR1下游一些新靶点，可能直接或间接调控细胞周期、线粒体和FASL/FAS凋亡通路。生物信息学显示p53在miR-149-5p宿主基因GPC1启动子区有潜在结合位点；突变型p53介导肿瘤耐药。由此我们提出科学假说：野生型和突变型p53差异转录GPC1和miR-149-5p，一方面通过CXCR1促进ERK和FAK/AKT通路，上调hnRNPK、Hsp27和Sorcin，介导线粒体和FASL/FAS凋亡通路crosstalk，导致细胞逃避化疗诱导的凋亡，另一方面GPC1和CXCR1通过crosstalk促进细胞周期；最终导致耐药。拟从细胞-动物-分子-临床四个层面验证假说。项目的完成将阐明p53通过GPC1/miR-149-5p介导胃癌耐药的新机制。

目前化疗耐药是胃癌治疗的瓶颈，是导致胃癌治疗失败的最主要原因之一。长期以来众多肿瘤学家及临床医生致力于胃癌的耐药机制的研究，以期寻找到新的治疗靶点。我们前期发现miR-149-5p靶向CXCR1；CXCR1促ERK和AKT磷酸化，介导胃癌耐药。前期蛋白质组学鉴定出CXCR1下游一些新靶点，可能直接或间接调控细胞周期、线粒体和FASL/FAS凋亡通路。生物信息学显示p53在miR-149-5p宿主基因GPC1启动子区有潜在结合位点；突变型p53介导肿瘤耐药。在本课题中，研究以下内容：（1）探讨野生型（wt）和突变型（mt）p53对miR-149-5p、宿主基因 GPC1以及 CXCR1的转录调控；（2）探讨 p53通过GPC1/miR-149-5p-CXCR1介导胃癌化疗耐药及其分子机制；（3）在临床组织水平证，探讨验证p53通过GPC1/miR-149-5p介导胃癌化疗耐药新机制。实验结果显示：在胃癌细胞中（1）由于 某些突变类型mt-p53（比如R248Q）蛋白DBD区三维空间构象的改变，mt-p53不能像wt-p53一样与miR-149-5p宿主基因GPC1启动子区的结合位点结合而启动GPC1转录，伴随GPC1 和 miR-149-5p 低水平或缺失，miR-149-5p 水平的下调致使其靶基因CXCR1失去抑制而表达上调；（2）高表达的 CXCR1与其配体结合激活ERK和FAK/AKT通路，从而介导线粒体凋亡通路和FASL/FAS凋亡通路“cross-talk”：共同抑制caspase-3活化，促使细胞逃避化疗药物诱导的凋亡；（3）p53/GPC1激活ERK和AKT通路与p53/miR-149-5p/CXCR1激活 ERK和FAK/AKT通路“cross-talk”；cyclin D1过表达，促进细胞周期；（4）细胞周期促进和凋亡逃避导致胃癌化疗耐药。项目阐明p53通过GPC1/miR-149-5p介导胃癌耐药的新机制，为胃癌的靶向治疗提供初步实验基础。