脾亢脾外泌体miRNAs诱导肝前体细胞损伤及EMT在肝癌发生中的作用及机制

The malignant transformation of hepatic precursor cells (HPCs) plays an important role in the development of hepatocellular carcinoma (HCC), but its mechanism is not yet clear. The hyperactive spleen (hypersplenism) is involved in the occurrence and development of liver cancer. Our previous study found that spleen and spleen excretion can induce cell damage and epithelial to mesenchymal transition (EMT) in HPCs. Exosomes can participate in the development of tumors by transmitting miRNAs. Therefore, we speculate that splenic anti-spleen miRNAs can promote the malignant transformation of HPCs by inducing cell damage and EMT, thereby promoting the occurrence of liver cancer. This project is planned to be based on the previous work: Firstly, in vitro and in vivo assays were used to detect changes in the malignant transformation ability of hypersplenism exosome-stimulated HPCs; secondly, exosomal miRNA sequencing, RNA-binding protein immunoprecipitation, and luciferase were used. Reporter genes and other methods screen out the miRNAs that play a major role in the spleen and spleen exosomes and verify their functions. Finally, the hypersplenism patients are analyzed for the differences in the abundance of splenic serum exosomal differential miRNAs and the pathological features of splenic fistula and liver cirrhosis. The correlation checks whether it can be used as a diagnostic marker. This project will provide a theoretical basis for studying the mechanism of hypersplenism excretion promoting the development of HCC.

肝前体细胞（HPCs）的恶性转化在肝细胞癌（HCC）发生中具有重要作用，但其机制尚不明确。功能亢进的脾脏（脾亢脾）参与肝癌的发生和发展。我们的前期研究发现，脾亢脾外泌体能诱导HPCs发生细胞损伤和上皮间质转化（EMT）。外泌体可以通过传输miRNAs参与肿瘤的发生发展。据此我们推测脾亢脾外泌体miRNAs可以通过诱导HPCs细胞损伤和EMT促进其恶性转化，进而促进肝癌的发生。本项目拟在前期工作基础上：首先利用体内体外实验检测脾亢脾外泌体刺激HPCs后其恶性转化能力的变化情况；其次，通过外泌体miRNA测序、RNA结合蛋白免疫沉淀、荧光素酶报告基因等手段筛选出脾亢脾外泌体中起主要作用的miRNAs，并验证其功能；最后，分析脾亢脾患者脾血清外泌体差异miRNAs的丰度与脾亢及肝硬化程度病理特征的相关性，验证其是否可以作为诊断标记。本项目将为研究脾亢脾外泌体促进HCC发生发展的机制提供理论基础。

肝前体细胞(HPCs)的恶性转化在肝细胞癌(HCC)的发生中具有重要作用，但其机制尚不明确。功能亢进的脾脏（脾亢脾）参与肝癌的发生和发展。在项目实施的三年中，我们围绕脾亢脾血清外泌体诱导HPCs损伤和EMT促进HCC发展的生物学作用和分子机制进行了系统性的研究。主要发现有1. 脾亢脾外泌体刺激后的HPCs发生细胞损伤。2. 脾亢脾血清外泌体刺激HPCs后细胞的干性减弱，出现部分抑制分化的特征。3. 脾亢脾血清外泌体长期刺激可以诱导HPCs 发生上皮间质转化EMT，表现为细胞形态从鹅卵石样变为梭状间质样、细胞间紧密连接消失、E-cadherin 等上皮细胞标志物表达下调、Vimentin 等间质细胞标志物表达成倍升高。4. 环转RNA Cdr1as在脾亢脾患者脾血清外泌体中特异高表达。5. 肝硬化小鼠脾脏Cdr1as的表达较对照组显著上调。6. Cdr1as可显著升高HPCs的增殖、克隆形成和侵袭能力，使HPCs产生肝癌肝细胞的部分特征。因此，我们的结果明确了脾亢脾血清外泌体在HPCs 细胞损伤恶性转化过程中的生物学作用，初步阐明了脾亢脾外泌体通过环状RNA Cdr1as诱导肝前体细胞恶性转化的新机制，为临床上治疗HCC提供了新的思路。上述部分成果已经在journal of Cellular of Biochemstry, Thoracic Cancer, International Immunopharmacology, Digestive and liver Disease等国际期刊发表研究论文共计4篇，其余研究结果目前正在整理投稿之中。