ZEB1-miR-300-FAK通路在肝癌上皮-间质转化中的作用及其机制研究

Epithelial-mesenchymal transition (EMT) is an important part of the initiating stage of the metastasis in liver cancer. Studies have shown that miRNA's regulatory effect on EMT. We found that low expression of miR-300 is closely associated with EMT of the liver cancer, but the molecular mechanism of low expression of the miR-300 and its function to the EMT is still unknown? We further found that: ZEB1 transcription factor whose expression may be downregulated by its action to the miR-300 promoter; miR-300 may inhibit the liver cancer EMT by the predicting target genes FAK. Therefore we hypothesized that: ZEB1 through inhibition of miR-300 and then lost its action to FAK promotes EMT of the liver cancer. To prove this hypothesis, this project intends to define the role of miR-300 in liver cancer EMT by in vivo and vitro experiments; confirme the specific role that the ZEB1's downregulatory function to the miR-300; identify the ZEB1-miR-300-FAK pathway's mechanism and action of the EMT in HCC; analysis the correlation between ZEB1, miR-300, FAK and EMT in large scale clinical HCC specimens; liver cancer and their significance on clinicopathological and prognostic. This will provide a reliable scientific basis for establishing miR-300 as an anti-HCC EMT targets for early diagnosis and treatment.

上皮间质转化（EMT）是肝癌转移的重要始动环节。研究显示miRNA对EMT具有调控作用。我们前期发现miR-300低表达与肝癌EMT发生密切相关，但miR-300对肝癌EMT的作用及其自身低表达的分子机制不明？我们进一步发现：ZEB1转录因子可能通过作用于miR-300启动子下调其表达；miR-300可能再通过预测靶基因FAK抑制肝癌EMT。故我们推测：ZEB1通过抑制miR-300进而失去对FAK的作用促进肝癌EMT。为论证该假说，本项目拟通过体内外实验明确miR-300对肝癌EMT的作用；证实ZEB1 下调miR-300具体作用；阐明ZEB1-miR-300-FAK通路在肝癌EMT中的作用及机制；分析大量临床肝癌组织标本中ZEB1、miR-300、FAK与EMT的相关性；以及它们对肝癌临床病理和预后的意义。这将为确立miR-300作为抗肝癌EMT的早期诊断和靶点治疗提供可靠的科学依据。

研究表明研究miR-300与肿瘤发生发展密切相关，但是其在肿瘤中的确切作用机制尚未明确。虽然目前miR-300在多种不同的肿瘤中均有研究报道，但是miR-300在肝癌中的具体作用机制尚未阐明。.本项目为了探究miR-300在肝癌中的具体作用机制，我们首先通过检测肝癌组织和细胞中的基础表达，结果发现miR-300在肝癌组织和肝癌细胞中普遍下调。结合临床资料分析发现，其表达下调肝癌预后不佳存在相关性。细胞功能实验进一步发现miR-300下调与肝癌的侵袭转移密切相关。随后通过实验发现miR-300在抑制EMT和肝癌转移中发挥重要的作用。并在机制上证明miR-300通过靶向调控粘着斑激酶（Focal adhesion kinase，FAK）抑制PI3K/AKT信号通路产生抑制EMT介导的肝癌侵袭和转移。使用Real Time PCR或者原位杂交的方法对肝癌临床组织样本和肝癌细胞中miR-300表达水平进行检测。Transwell迁移和侵袭实验用来评估miR-300过表达或敲低后肝癌细胞的体外侵袭能力。Real Time PCR、Western Blotting、免疫荧光和免疫组织化学等方法用来验证miR-300和FAK/PI3K/AKT在EMT进展中的作用。双荧光素酶报告实验用来确定miR-300的靶基因。双荧光素酶报告试验和拯救实验证实了miR-300通过靶向FAK和下游的PI3K/AKT信号通路来抑制EMT和肝癌侵袭转移。本项目的研究结果表明miR-300有可能是肝癌治疗的新靶点。