基于ceRNA调控网络研究复方鳖甲软肝片对合并肝纤维化肝癌的干预机制

Hepatic fibrosis may lead to cirrhosis, which may progress to liver cancer, with an associated risk of liver failure and death. Effective anti-fibrosis treatment is of great significance for the prevention of liver cancer. Emerging clinical evidence suggests that traditional Chinese medicine (TCM) formula Fufang Biejia Ruangan Pills (FBRP) may be an efficient agent to attenuate the severity of liver inflammation and hepatic fibrosis, and improve the immune state of the whole body. However, its potential clinical applications and underlying mechanisms acting on hepatocellular carcinoma (HCC) with a hepatic fibrosis background remain unclear. To fill this gap, microarray detection of mRNAs, long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) will be performed in this project using human hepatic fibrosis and HCC tissues. Following the bioinformatic analyses on RNA expression profiles and the construction of lncRNA-miRNA-mRNA network, the candidate mRNAs/miRNAs/lncRNAs/competing endogenous RNAs (ceRNAs) associated with hepatic fibrosis-HCC axis will be identified at a system level. Then, the putative targets for composite compounds contained in FBRP will be predicted using a TCM target prediction system which was established by our previous studies. After that, the drug target--disease-related RNA interaction network will be constructed and a list of major FBRP targets acting on HCC will be generated based on their topological features and their links to the candidate mRNAs/miRNAs/lncRNAs/ceRNAs associated with hepatic fibrosis-HCC axis. Further experimental validations in vivo and in vitro systems will be performed to verify our prediction results. This study will not only enrich the understanding of the process from hepatic fibrosis to HCC, but also offer a powerful evidence for the potential clinical applications of FBRP. It is helpful to broaden the scope of FBRP's clinical applications, as well as provide novel theoretical basis and therapeutic strategies for the comprehensive prevention and treatment of human HCC.

肝纤维化是肝炎发展为肝硬化、肝癌的中间环节，有效的抗肝纤维化治疗对于肝癌的防治具有重要意义。抗肝纤维化首选中成药复方鳖甲软肝片在抗炎、抗纤维化及增强机体免疫功能等方面具有明显优势。然而，该方对于肝癌的干预特点和作用原理尚不清楚。本项目拟在前期工作基础上，基于肝纤维化和合并肝纤维化肝癌的临床样本，整合mRNA/miRNA/lncRNA多分子层面芯片和网络分析，系统鉴定肝纤维化-肝癌轴相关RNAs及竞争性内源RNA（ceRNA）组合；再利用本课题组已建立的中药靶标预测系统，获得复方鳖甲软肝片的候选靶标谱，并通过疾病相关RNAs/ceRNAs及药物靶标互作网络分析和实验验证，探索该方对肝纤维化-肝癌轴相关ceRNA调控网络的干预机制，明确其对合并肝纤维化肝癌的治疗作用。本项目将揭示复方鳖甲软肝片防治肝癌的潜能，有利于拓宽该方的应用范围及研究思路，也为肝癌的综合治疗提供新的理论基础和干预手段。

针对复方鳖甲软肝片（FBRP）缓解肝脏“炎-癌”恶性转化的作用特点和分子机制尚不明确的科学问题，本项目从临床收集肝纤维化合并肝癌及非癌肝纤维化组织样本，通过全转录组检测，获得与肝纤维化-肝癌恶性轴相关的mRNA、miRNA和lncRNA差异表达谱。接着，通过构建和分析“疾病差异基因-药物候选靶标”互作网络，发现FBRP组方消癥散结、补血活血、益气健脾、育阴养肝和清热解毒等5个主要功效组的核心靶标群除了均可干预癌症相关的炎症-免疫失衡、物质和能量代谢失调等通路，也作用于不同功效组特有的通路群，一方面增强免疫功能、干预细胞周期，以消除癌症对机体不利的因素，另一方面改善神经和代谢异常，以推动机体恢复动态平衡，这与FBRP中医组方“标本兼顾、消补兼施”的理论相契合。进一步，从FBRP候选靶标与肝纤维化-肝癌恶性轴相关基因互作网络出发，通过关键网络靶标识别和功能模块分析，发现PI3K/Akt/NF-κB信号介导的相关途径可能是FBRP逆转肝纤维化-肝癌轴的候选核心靶点，通过体内、体外系列实验，验证了FBRP逆转肝脏“炎-癌”恶性转化的药效及其调控PI3K/Akt/NF-κB信号转导的作用。然后，利用前期临床样本来源的mRNA、miRNA和lncRNA差异表达谱，开展miRNA靶基因和靶lncRNA预测、miRNA-靶mRNA和miRNA-靶lncRNA共表达网络分析、及“临床-动物-细胞”多层次实验，明确了LncRNA TUG1-miR-328-3p-SRSF9 ceRNA调控轴的失调与肝纤维化-肝癌恶性轴密切相关，及FBRP对该ceRNA调控轴的干预作用。本项目在理论分析FBRP抗肝癌潜能及其组方功效配伍特点的基础上，结合转录组学检测、网络挖掘和实验验证，不仅考察了FBRP干预肝癌发生和进展的药效，也探明了该方对肝“炎-癌”恶性转化相关ceRNA调控网络的干预机制，为拓宽其临床应用范围提供新的理论基础和实验证据。