microRNA分子参与肿瘤细胞上皮-间质转化过程中鲁棒性维持的分子机制

Biological robustness is a system property to keep a stability of live-body structure and function as uncertainty factor from external and internal variety disturbs it. Biological systems use a variety of mechanisms to maintain their functions in the face of environmental and genetic perturbations. Increasing evidence suggests that, among their roles as posttranscriptional repressors of gene expression, microRNAs (miRNAs) help to confer robustness to biological processes by reinforcing transcriptional programs and attenuating aberrant transcripts. These activities have important consequences for normal development and physiology, disease, and evolution. The majority of deaths from cancer are caused by the tumor invasion and metastasis. Epithelial-mesenchymal transition (EMT) is critical for appropriate embryonic development, and this process is re-engaged in adults during wound healing, tissue regeneration, organ fibrosis, and cancer invasion and metastasis. EMT can be prompted by various intrinsic signals (e.g. gene mutations) as well extrinsic signals (e.g. growth factor signaling). Among the growth factors known to induce EMT are transforming growth factor β (TGF-β) and epidermal growth factor (EGF) family. We have used TGF-β and EGF to induce nasopharyngeal carcinoma (NPC) cell to undergo EMT, and profiled the mRNA and miRNA expression pattern changes in this process. We discovered a few of miRNA related feedback and feedforward loops involving in the EMT process. We observed an upregulation of miR-505, and miR-1207-5p upon EMT, and found that all of them could suppress tumor cell migration and invasion abilities, conferring robustness to the EMT process. In this proposal, we will systematically investigate the downstream target genes of miR-505 and miR-1207-5p in the EMT process; evaluate whether their inhibition of EMT depend on their ability to suppress TGF-β and Wnt/β-catenin signaling; explore their roles of controlling the TGF-β-induced "EMT or apoptosis switch" and their roles in the tumor microenvironment. The illumination of the microRNA's role in conferring robustness to EMT process may help us to understand their function in preventing cancer invasion and metastasis, and provide new possibility to discover useful therauptic targets for cancer metastasis.

生物鲁棒性是生物体在内外界环境变化时维持其结构和功能稳定的一种特性，miRNA分子作为基因表达的精细调控者参与维持生物鲁棒性。我们分析了EGF或TGF-β诱导鼻咽癌细胞发生上皮-间质转化(EMT)时mRNA和miRNA表达谱变化，构建了miRNA参与维持EMT过程中鲁棒性的几个重要反馈环路。发现EMT过程中表达上调的miR-505、-1207-5p能抑制癌细胞迁移和侵袭能力，负调控EMT，维持系统鲁棒性。本项目中我们拟系统鉴定miR-505和miR-1207-5p在EMT过程中的靶基因；分析它们抑制EMT的机制是否与TGF-β和Wnt/β-catenin通路相关；它们对TGF-β诱导的"EMT-凋亡转换"的调控；以及它们在肿瘤微环境中如何发挥作用。这些研究将有助于更好理解EMT过程的鲁棒性，以及miRNA调控肿瘤侵袭转移的分子机制，为肿瘤转移的防治提供新的靶点。

我们研究了EGF或TGF-β诱导鼻咽癌细胞发生上皮-间质转化(EMT)时miRNA表达谱变化，发现了miR-148a、-505、-1207-5p、-21分子表达上调。miR-21是著名的癌基因miRNA分子，因此我们最初推测：miR-148a、-505、-1207-5p也是具有癌基因功能的miRNA分子，促进EMT。但是在实际的研究过程中，我们发现，miR-148a、-505、-1207-5p都在EMT过程中发挥负调控作用，抑制EMT过程，说明这些EMT过程中被诱导的microRNA分子，发挥了“缓冲剂”的作用，维持EMT过程中的鲁棒性。这个发现在既往的研究中是较少被关注的。.我们确认了AMFR是miR-148a的直接靶基因；CD-151和CSF1是miR-1207–5p的直接靶基因。CSF1对于巨噬细胞的生成、分化、功能都起到重要的促进作用，是肿瘤微环境的一个重要因素。miR-1207-5p对于CSF1的靶向抑制作用，揭示该分子能够调控肿瘤微环境，抑制肿瘤转移。这三个miRNA分子能抑制肿瘤细胞在裸鼠体内的转移；显著降低鼻咽癌细胞中侧群细胞的比例，减弱肿瘤微球的形成能力。通过PTEN/PDK1/AKT/GSK3β/β-catenin调控轴线上调β-catenin的磷酸化水平，负调Wnt信号通路。上调Axin1的蛋白表达水平则是它们负调Wnt通路的另一途径。这三个miRNA分子能够通过多种途径负调Wnt信号通路从而抑制肿瘤细胞的EMT进程和肿瘤干细胞活性。我们还进一步发现这三个microRNA分子可以显著抑制具有促癌功能的MALAT1、HOTAIR、BCAR4等lncRNA的表达。MALAT1介导了三个miRNA分子对Wnt通路的负调控作用。.除此之外，我们鉴定出了一个新的细胞密度敏感性的，且能靶向抑制Hippo通路YAP1的miRNA分子：miR-590-5p。我们发现了一个受到YAP1诱导的lncRNA分子：LINC00152，揭示 “LINC00152/miR-632-miR-185-3p/FSCN1”轴对结直肠癌细胞增殖、侵袭转移的恶性生物学行为。.本项目整体研究内容充实，充分揭示了miRNA分子对于维持EMT鲁棒性的机制和生理学意义；发现了参与YAP1通路调控的新的非编码RNA分子，对于解释细胞密度-肿瘤生物学行为具有较重要的价值。.