MiR-200a缺失后调控EMT过程参与肝干细胞恶性转化的分子机制研究

One of the most recognized mechanisms for liver cancer stem cells (LCSCs) genesis is from the malignant transformation of liver normal stem cells (LNSCs), which gain multiple genetic changes under the stimulation of cancer inductive factors. However, the exact mechanisms for this malignant transformation is so far unclear. In order to explore some possible molecular mechanisms, we isolated both LCSCs and LNSCs using our established methods, and compared their microRNA and genetic expression profiles. Among a great number of dys-regulated microRNAs and genes, we selected the most down-regulated molecule miR-200a (almost lack of expression in LCSCs) for later thorough exploration. On the support of our previous study, we found that the inhibition of miR-200a could push LNSCs to proliferate at much fast speed and easily migrate. As a result, through the above changes, the inhibition of miR-200a could finally induce LNSCs to go through malignant transformation. Although the above mentioned findings have been sequentially published in several professional journals including "Stem Cell Rev", the detailed molecular mechanism responsible for the malignant transformation of LNSCs induced by the miR-200a inhibition is still unclear. A number of recent researches reveal that the biological functions of miR-200a are mainly accomplished by regulating an important biological event, epithelial-mesenchymal transition (EMT). This project will follow the preliminary findings, to further confirm the contribution of EMT in the miR-200a inhibition induced LNSCs malignant transformation, explore the molecular mechanisms for miR-200a regulating each step of EMT, and eventually clarify the molecular mechanisms that miR-200a inhibition leads to the malignant transformation of LNSCs.?Through these researches, it will not only expand the knowledges of miR-200a-inhition related molecular mechanisms, but also provide novel targets for preventing the malignant transformtion of LNSCs.

最近研究表明，肝干细胞可恶性转化为肝癌干细胞，进而引起肝癌发生、发展，但其中的分子机制不清楚。我们前期建立了分离肝干细胞和肝癌干细胞的两种方法，并在此基础上获得了两种干细胞间miRNA和基因差异表达谱；进一步针对最大差异表达分子miR-200a的研究发现，肝干细胞中抑制其表达会促进细胞增殖加快和侵袭迁移，呈现类似肝癌干细胞的特征。虽然miR-200a缺失后诱导肝干细胞恶性转化的现象得到证实，但是其中的关键分子机制仍不清楚。已有研究提出，miR-200a的生物学功能可能主要是通过调控上皮-间充质转化（EMT）加以实现的。本项目将在我们前期研究的基础上，重点探索miR-200a缺失后如何调控EMT发生的各个环节，以此为切入点阐明miR-200a缺失后促进肝干细胞恶性转化的关键分子机制。这些研究不仅可以发现阻止肝干细胞向肝癌干细胞恶性转化的新靶点，而且可以拓展对miR-200a调控机制的新认识。

最近研究表明，肝干细胞可恶性转化为肝癌干细胞，进而引起肝癌发生、发展，但其中的分子机制不清楚。本研究初步阐明了miR-200a缺失表达后诱导肝干细胞恶性转化的相关分子机制，即miR-200a缺失表达后能够造成EMT过程的发生。首先，我们证实抑制miR-200a的表达后，肝干细胞系WB-F344呈现间充质细胞特征，包括细长的细胞形态、增强的迁移能力、高表达EMT的M期标志物、呈现肿瘤干细胞的标志物。 进一步地，通过双荧光素酶报告系统等方案，我们证实miR-200a缺失后会导致期靶向调控的基因beta-catenin (CTNNB1) 高表达，可能是其调控EMT的分子机制。我们还对比了101例原发性肝癌组织和其配对的正常肝脏组织中miR-200a的表达水平，并分析了miR-200a表达丰度与临床病理之间的关系。结果发现，相对于正常肝组织，miR-200a在肝癌组织显著低表达，尤其在原发性肝癌伴转移的病例中几乎缺失表达。此外，通过建立的肝癌干细胞分选方法，我们从肝癌细胞系中分选出肝癌干细胞亚群（side population, SP）及肝癌非干细胞亚群（non side population, NSP），比较了两者的miR-200a表达情况。通过对比发现，SP细胞较NSP细胞低表达miR-200a， 同时具有低表达EMT的E期标志物E-cadherin及高表达M期标志物N-cadherin。鉴于miR-200a在其它研究中均证实其能调控EMT过程，我们在SP细胞中转染了miR-200a的模拟物，结果发现，M期标志物N-cadherin 、vimentin及ZEB2 显著下调，E期标志物E-cadherin明显上调。进一步的研究发现，miR-200a可能通过直接靶向调控转录因子ZEB2，进而调控着EMT过程。通过以上研究，我们初步揭开了miR-200a调控EMT的分子机制，为阻止肝干细胞向肝癌干细胞恶性转化提供了新靶点。