CIRP调控Tim-3介导NK功能抑制在肝细胞肝癌进展中的作用及机制

Overexpression of cold inducible RNA-binding protein (CIRP) could promote the proliferation of tumor cells. Extracellular secretory CIRP could bind to TLR4 and further induce the inflammatory response, but the relationship between secretory CIRP and cancer is unclear. In previous study, we firstly discovered that the level of serum CIRP in hepatocellular carcinoma (HCC) patients was significantly elevated, which was negatively correlated with prognosis. Moreover, CIRP protein inhibited the activity of NK cells and induced Tim-3 expression in vitro. Previous studies have shown that TLR4 ligand could induce the expression of Tim-3, and the activation of TLR4 downstream MAPK signaling pathway promoted the transcription of Tim-3. Therefore, we proposed that secretory CIRP induced Tim-3 expression by activating the TLR4-MAPK signaling pathway, thereby enhancing the Tim-3 mediated inhibition of NK function and ultimately promoting the progression of HCC. In the further study, we intend to use Tim-3 blocking antibody, lentivirus infection, signal pathway inhibitors and siRNA transfection in vitro and in the mouse model of HCC in vivo to investigate. This project is expected to reveal the novel mechanism of secretory CIRP in promoting the development of HCC, and would provide a theoretical basis for clinical screening of new targets for cancer diagnosis and treatment.

冷诱导RNA结合蛋白（CIRP）在胞内过表达促进肿瘤生长，分泌至胞外的CIRP与TLR4结合可介导炎症反应，但分泌型CIRP与肿瘤的关系目前尚不清楚。申请人前期首次发现，肝细胞肝癌（HCC）患者血清中CIRP表达明显升高，并与预后显著负相关。同时，CIRP蛋白体外抑制NK功能并上调负性调控分子Tim-3的表达。既往研究表明，TLR4配体能够上调Tim-3表达，并且TLR4下游MAPK信号通路的激活能够促进Tim-3的转录。因此,我们提出假设：分泌型CIRP通过激活TLR4-MAPK信号通路上调Tim-3表达，从而增强Tim-3介导的NK功能抑制，最终促进HCC进展。本项目拟计划以NK细胞为核心，在细胞体外及小鼠肝癌体内模型上，采用Tim-3阻断抗体、慢病毒感染、信号通路抑制剂、siRNA转染等技术，揭示分泌型CIRP促进HCC进展的全新机制，并为临床筛选诊断及抗癌新靶点提供理论依据。

肝细胞肝癌（hepatocellular carcinoma，HCC）是恶性程度高，进展快，病死率高。深入研究肝细胞肝癌发生和发展过程的调控新机制，找到有效的诊断和预后标记物及干预靶点，成为HCC研究的当务之急。.冷诱导RNA结合蛋白（cold inducible RNA binding protein, CIRP/CIRBP）是在哺乳动物细胞中被确定的第一个冷休克蛋白，其在胞内过表达促进肿瘤生长，分泌至胞外的CIRP与TLR4结合可介导炎症反应，但分泌型CIRP与肿瘤的关系目前尚不清楚。.本项目研究发现：1. 肝细胞肝癌（HCC）患者血清中CIRP表达明显升高，并与预后显著负相关；2. TCGA数据库分析并结合肝癌组织芯片免疫组化染色证实CIRP在肝癌组织的中的表达显著高于癌旁；3. CIRP体内促进H22荷瘤鼠肿瘤的生长，而其阻断剂抑制CIRP诱导的促进肿瘤生长的作用; 4. 对肿瘤组织浸润淋巴细胞的百分比进行检测，发现与模型组相比，CIRP注射组NK细胞百分比下降，体外实验进一步证实CIRP可直接抑制NK细胞活性；5.对实验组荷瘤鼠肿瘤组织中的免疫检查点基因TIM-3表达上升，进一步肝癌组织芯片染色证实CIRP表达与TIM-3表达正相关；6. Anti-TIM-3抗体阻断逆转CIRP抑制NK细胞活性；7. CIRP 上调肝癌细胞TIM-3表达，且呈时间及剂量依赖性；8. 采用anti-TLR4抗体阻断、MyD88抑制剂结合western-bolt证实CIRP通过TLR4/MyD88/MAPK信号通路上调TIM-3表达；9. siRNA沉默STAT3结合双荧光素酶报告基因实验证实，CIRP通过激活STAT3进而促进TIM-3转录。.本项目研究揭示了分泌型CIRP促进HCC进展的全新机制，并为临床筛选诊断及抗癌新靶点提供理论依据。