力学应答microRNAs在肝癌分化与侵袭中的作用及其机制

The pressure in hepatic microenvironment plays an import role in the development and metastasis of liver cancer. However, the mechanism of stretch in this is unclear. In our previous research, some microRNA were screened and validated under different pressure and strech in hepatic fibrosis models. And we also studied on the role of hepatic nuclear factor 4α (HNF4α) in hepatic differentiation and metastasis of hepatic carcinoma. In this study, hepatocytes from hESCs or hepatic cancer cells will be exposed to different pressure and stretch using a pressure inducing apparatus and programmed cell traction device Flexercell Strain Unit 2000 respectively. Using the microRNA microarray approach, we will compare the microRNA expression profiles following proper pressure or stretch. Then recombinant lentivirus encoding biomechanics responsive microRNAs will be constructed and their effects on hepatocytes or hepatic cancer cells responding to biomechanics will be studied. Furthermore, the expression of target genes and microRNAs will be validated in animal models and from clinical data. This study helps to better understand the pathogenesis of hepatic carcinoma.And we aim to regulate the expression of microRNA to control the fate of hepatocyte differentiation and proliferation under different mechanical conditions and further investigate how microRNA regulate the activation and role of HNF4α in hepatic carcinoma, which will provide a new way for the treatment of liver cancer and finding effective drugs.

肝脏力学微环境变化及引起的肝硬化可能参与肝癌发生和转移的调节，但其机制尚不清楚。我们前期完成了不同应力条件下肝硬化miRNA的筛选和鉴定，并对影响肝癌细胞分化和转移的重要因子HNF4α进行了初步研究。本项目拟在前期工作基础上，模拟肝硬化时肝内力学微环境条件，分别对诱导分化肝细胞和人肝癌细胞株进行静水压力和机械张应变干预，然后筛选力学应答的差异表达miRNA 并进行生物学验证；建立裸鼠原位肝癌转移模型，构建力学应答miRNA过表达或低表达载体观察可能的疗效；并通过临床资料验证力学应答的miRNAs表达；明确不同力学条件下，可调控肝癌细胞分化和转移的力学应答miRNA的差异表达谱，探讨力学应答miRNA对肝癌细胞分化和转移的重要因子HNF4α的调控作用，为肝癌的预防和治疗提供力学生物学理论依据，并为寻找临床治疗肝癌的有效药物提供方向。

肝脏力学微环境变化可能参与肝癌发生和转移的调节，本项目模拟肝硬化时肝内力学微环境条件，分别对肝细胞和人肝癌细胞株进行静水压力和机械张应变干预，发现15mmHg，24h压力加载显著促进肝癌细胞增殖、迁移和侵袭，利用芯片筛选出5个压力应答的差异表达 miRNA：miRNA-630、miRNA-5703表达下调，miRNA-7641、miRNA-4485-3p、miRNA-7-5p表达上调；而张应变幅度15%、频率1Hz、时间24h加载可促进肝癌细胞增殖，筛选出7个张应变力应答miRNA：miRNA-296-5p、miRNA-6752-5p、miRNA-6794-5p、miRNA-6889-5p、miRNA-7845-5p上调，miRNA-503-5p和miRNA-4428下调。经GO分析和Pathway分析确定压力应答miRNA-5703为本实验研究的目的miRNA，预测靶基因为SRC。构建miRNA-5703过表达及SRC干扰慢病毒，利用双荧光素酶报告基因验证靶向关系；通过Western-blotting实验观察压力下Focal adhesion通路、PI3K通路和FOXO通路相关蛋白以及paxillin的表达变化；建立裸鼠皮下荷瘤模型，发现过表达miRNA-5703的肿瘤重量明显低于对照组，说明miRNA-5703可以明显抑制压力下肝癌的增殖；临床收集BCLC分级为0级和A1级（无门脉高压）的患者的肝癌组织及其成对癌旁正常组织22对，以及分级为A2-D级的25对。通过RT-qPCR实验检测miRNA-5703和Src的表达。结果显示无门脉高压患者的组织中，20对miRNA-5703在肝癌组织和癌旁正常组织中的表达差异倍数在0.9到1.1之间，17对SRC在肝癌组织中的表达高于癌旁正常组织，其中9对表达小于2倍；有门脉高压的患者组织中，19对miRNA-5703在肝癌组织中的表达低于癌旁正常组织，其中10对miRNA-5703表达<0.5倍，21对肝癌组织中SRC表达高于癌旁正常组织，13对表达>2倍。本课题明确了不同力学条件下，可调控肝癌细胞增殖和转移的力学应答miRNA的差异表达谱，同时证明了压力响应miRNA-5703可以通过下调Src表达抑制压力下肝癌的生长和转移，为肝癌的预防和治疗提供力学生物学客观依据，并为寻找临床治疗肝癌的有效药物提供方向。