PD-1是调节肿瘤和免疫的关键蛋白之一，然而目前对其表达调控的分子机制尚不完全清楚，这阻碍了对肿瘤发生和肿瘤免疫机制的认识。我们最近的前期实验结果显示，PD-1 mRNA的3’UTR对基因表达有强烈的抑制作用。进一步研究发现它能在转录后水平显著促进mRNA降解，而其调节活性依赖于3’UTR上一个特殊的茎环二级结构。为进一步阐明其分子作用机制，本项目拟采用生物信息分析、Aptamer亲和纯化等方法鉴定与茎环结构元件相互作用的RNA结合蛋白（RBP）；然后利用基因敲除、荧光报告实验、流式细胞分析等手段弄清RBP与结构元件相互作用对PD-1 mRNA降解速率和蛋白水平、以及对细胞增殖的影响和机制；进一步将采用Co-IP等方法揭示RBP招募去腺苷酸酶以去除polyA尾巴而加速mRNA降解的过程。该研究有望加深对基因表达调控复杂性的认识，揭示PD-1表达调控的新机制，为探索肿瘤治疗的新策略提供基础。

PD-1 is a crucial protein for regulation of tumorigenesis and immunity. However, the mechanism on which the PD-1 expression is regulated remains largely unknown, which is hindering us from understanding how T cells and cancer cells are mechanistically regulated. We recently used a fluorescence reporter assay to demonstrate that the 3’UTR of PD-1 mRNA possesses strong activity in inhibition of PD-1 expression, which was further supported by the results from a CRISPR based in vivo assay. Additionally, we found that the 3’UTR regulated gene expression at post-transcriptional level by destabilizing mRNA. Truncation test showed that the destabilizing activity of PD-1 3’UTR depended on a specific region that can form a secondary step-loop structure. To elucidate the molecular mechanism on which the step-loop structure contributes to the 3’UTR regulatory activity, we plan to use bioinformatic analysis and Aptamer affinity purification to identify the RNA binding protein (RBP) that interacts with the stem-loop structure. Next we will study the effect of the interaction between RBP and the stem-loop structure on PD-1 mRNA degradation, protein output, and cellular proliferation by using a set of methods including gene knock-out, fluorescence reporter assay, and flow cytometric analysis. Further, we will utilize Co-IP method to reveal the process where the RBP recruits the deadenylase complex that hypothetically removes the polyA tail from mRNA and accelerates mRNA degradation. We expect that this study would deepen our understanding of the complexity of gene expression regulation, and reveal a new mechanism for regulating PD-1 expression, which could potentially provide new options for developing new methods for cancer treatment.