蒙药德都红花七味丸调控miR-23a/DAPK1/PVT1轴的抗肝癌机制研究

Primary hepatocellular carcinoma (HCC), as a common malignant tumor in clinical practice, still has unclear pathogenesis and poor prognosis currently. HCC belongs to “malignant fatty liver diseases” in traditional Mongolian medicine that uses Mongolian medicine Dedu Pills of 7 Drugs with Safflower to treat fatty liver and liver heat (removal of “Xieri” and dispelling of “Xila”). We have verified that Dedu Pills can significantly down-regulate miR-23a level of HCC HepG2 cells. In this project, based on the theory of traditional Mongolian medicine, we constructed DAPK1 and PVT1 plasmids to transfect cell lines through stable expression/knockout. The regulatory effects of miR-23a on DAPK1 in the presence of Dedu Pills were evaluated by using real-time fluorescent PCR, chromatin coprecipitation assay and luciferase reporter gene assay. Besides, DAPK1/miR-23a-regulated role of DAPK1 in HCC cell proliferation and invasion as well as the mechanisms were studied, providing evidence for clarifying the targeted regulatory effects of lncRNA on the miR-23a/DAPK1/PVT1 network. Moreover, the mechanisms by which Dedu Pills regulated PVT1 and miR-23a in human HCC cells to participate in the onset and progression of HCC were preliminarily studied. This project, for the first time, employs PVT1 and DAPK1 as the potential molecular targets for developing natural traditional Mongolian medicines. The cell-cycle specificity of Dedu Pills paves a way for developing analogous drugs that block tumor cell proliferation.

原发性肝癌病因复杂，现有治疗手段预后较差。蒙药德都红花七味丸在传统蒙古医学中主治肝痞热病，本课题组以往研究表明德都红花七味丸可显著下调肝癌HepG2细胞miR-23a水平。本项目通过构建DAPK1、PVT1等质粒稳定表达转染细胞株，运用qPCR、染色质共沉淀、荧光素酶报告基因等不同方法验证德都红花七味丸作用下miR-23a对DAPK1和PVT1的调控作用；在体外及体内水平探讨德都红花七味丸调控miR-23a/DAPK1/PVT1轴影响肝肿瘤血管生成和转移的机制；进而研究DAPK1和PVT1的互作关系及miR-23a对该通路的靶向调控作用。本研究拟阐明蒙药德都红花七味丸调控人肝癌细胞相关Lnc-RNA和miRNA在肝癌发生、发展中可能的作用机制；为以PVT1和DAPK1为潜在分子靶点的抗癌复方蒙药研发提供实验依据，初步阐明红花七味丸的抗癌机理可为后续开发类似的抗肿瘤药物提供方向。

原发性肝癌病因复杂，现有治疗手段预后较差。蒙药德都红花七味丸在传统蒙古医学中主治肝痞热病，课题组以往研究表明德都红花七味丸可显著下调肝癌HepG2细胞miR-23a水平。在本国自然项目中，项目组通过构建DAPK1、PVT1等质粒稳定表达转染细胞株，运用qPCR、染色质共沉淀、荧光素酶报告基因等不同方法验证德都红花七味丸作用下miR-23a对DAPK1和PVT1的调控作用；在体外及体内水平探讨德都红花七味丸调控miR-23a/DAPK1/PVT1轴影响肝肿瘤血管生成和转移的机制；进而研究DAPK1和PVT1的互作关系及miR-23a对该通路的靶向调控作用。同时我们在研究中发现肝细胞癌及癌前肝纤维化中lncRNA LOC102551149和 miR-23a-5p之间的联系。并对miR-23a-5p进行了深入的研究，采用qRT-PCR技术在N-亚硝基二甲胺（NDMA）诱导的大鼠肝纤维化和血小板衍生生长因子（PDGF）诱导的HSC活化中检测miR-23a-5p。 经过H＆E染色，masson染色和剪切波电描记法（SWE）检测肝纤维化水平。通过免疫组织化学染色，qRT-PCR和western-blot检测肝纤维化或HSC激活的相对标记和相对途径基因及蛋白质。双荧光素酶报告系统验证了HSC-T6中miR-23a-5p与PTEN或miR-23a-5p与lncRNA LOC102551149之间的相互作用。 siRNA和miRNA模拟转移到HSC-T6，检测lncRNA LOC102551149和miR-23a-5p对HSC激活的作用。结果发现：肝纤维化和HSC激活发生后，miR-23a-5p上调，而抗miR-23a-5p可以使肝纤维化和HSC激活恢复。进一步的研究表明，miR-23a-5p可以靶向PTEN并将其降解，从而引起PI3K / Akt / mTOR / Snail途径的激活。 lncRNA LOC102551149可通过碱基配对充当靶向miR-23a-5p的竞争内源性RNA（ceRNA），而siRNA LOC102551149或外源性miR-23a-5p可通过PI3K / Akt / mTOR / Snail途径诱导HSC激活。通过以上研究我们证明了miR-23a-5p对肝纤维化和HSC活化的作用途径，可能为肝癌及纤维化的治疗提供靶点。