内源性miRNAs在血小板介导的肝癌细胞免疫逃逸及血管定植中的关键作用及机制研究

Tumor metastasis and recurrence is the major prognosis determinant of postoperative hepatocellular carcinoma.Researches have shown that platelets play key auxiliary role in tumor recurrence and metastasis, but its exact mechanism is still unclear.Recent studies have found that the platelet miRNAs is an important part in platelet function regulating mechanism. According to our previous results of researches on platelets and hepatocellular cancer recurrence and metastasis, we bring up our assumption that platelet-derived miRNA is likely to participates in hepatocellular carcinoma key step of metastasis and recurrence, such as cancer cells' immune evasion and microvascular implantation, by accommodate platelet function. Based on this, the project intends to:①Clarify the mode of platelets involving in metastasis of hepatocellular carcinoma cell depend on selected clinical cases and cell models; ②Screening platelets endogenous miRNAs closely related to hepatoma cell metastasis and platelet function by high-throughput microarray and bioinformatics analysis;③Explore and clarify the mechanisms of platelet-derived miRNAs participate in by the platelet-mediated hepatoma carcinoma cell's immune evasion and microvascular implantation in cell and animal models;④Explore the feasibility of platelet-derived miRNA as a novel marker of HCC by transformating results of basic research.

肿瘤复发转移是决定肝癌术后疗效的主要因素。研究表明，血小板在肿瘤复发转移中发挥关键性辅助作用，然而其具体机制尚不明确。最近研究发现，血小板内miRNAs是调控血小板功能的重要机制。课题组前期在大量血小板与肝癌复发转移相关研究基础上提出，血小板内源性miRNA极可能通过影响血小板功能，参与肝癌细胞免疫逃逸及血管定植等关键复发转移步骤。基于此，本项目拟：①选择临床病例及细胞模型，明确血小板参与肝癌细胞转移的作用模式；②利用高通量芯片及生物信息学分析，筛选影响肝癌细胞转移的与血小板功能密切相关的内源性miRNAs；③通过细胞及动物模型，阐明血小板内源性miRNAs对由血小板介导的肝癌细胞免疫逃逸及血管定植的调控机制；④转化基础研究成果，初步探讨血小板内源性miRNA作为肝癌新型标志物可行性。本项目的完成将进一步拓展对血小板参与肝癌复发转移机制的研究领域，为建立肝癌早期监测及治疗的新策略提供思路。

肿瘤转移是制约肝癌患者预后的关键因素之一。既往有研究证明血小板在肿瘤转移和复发过程中发挥关键性作用，然而其在肝癌中的具体机制目前尚无研究。本课题收集肝癌患者临床资料与血小板计数进行相关分析，以离心-磁珠分选技术为基础体外纯化血小板，体内及体外实验分别探索血小板对肝癌细胞多种生物学行为的影响，通过血小板黏附实验及免疫逃逸途径的标志物检测明确血小板对肝癌细胞免疫逃逸及血管定植的作用。对肝癌患者和健康志愿者的血小板进行新一代微流体miRNA芯片测序结果结合生物信息学分析技术筛选肝癌特异性血小板关键miRNAs,对其在肝癌转移过程中参与免疫逃逸的功能进行研究。项目实验结果证实高血小板计数是影响肝癌不良预后的独立危险因素，活化的血小板具有更强的与肿瘤细胞及血管内皮细胞黏附作用，可以通过调控Fas/FasL途径影响肝癌细胞的免疫逃逸从而促进肝癌细胞的增殖转移能力；miRNA测序筛选得到miR-34a、miR-1271和miR-146b-5p在体内和体外均可以促进肝癌细胞的增殖转移并且影响Fas/FasL介导的肝癌免疫逃逸，并且miR-34a、miR-1271和miR-146b-5p能够作为预测肝癌复发转移指标预测肝癌患者的肿瘤转移情况。结论：血小板通过促进肝癌细胞的免疫逃逸和血管定植影响肝癌转移，影响肝癌患者预后；多种血小板关键miRNAs共同影响肝癌细胞的侵袭转移能力。本项目的完成将进一步拓展对血小板参与肝癌复发转移机制的研究领域，为建立肝癌早期监测及治疗的新策略提供思路。