c-Met和DLK1-DIO3印记域miRNA簇之间的调控环路在膀胱癌恶性行为中的作用及其机制研究

Urinary bladder cancer is the most common urogenital malignant tumor. To elucidate the molecular mechanism of bladder cancer is important for seeking tumor markers and therapeutic targets. In our previous research, we found that c-Met was overexpressed in bladder cancer, whereas more than 50 miRNAs within the DLK1-DIO3 genomic region was down-regulated. In addition, there appears to exist a reciprocal regulation between them. Therefore, our current study intends to explore the epigenetic and transcriptional regulation role of c-Met in miRNAs within the DLK1-DIO3 genomic region. We would also try to identify the miRNAs within the DLK1-DIO3 genomic region that could directly regulate the expression of c-Met. Finally, we will adopt a novel endogenous gene activation technique, namely CRISPR-on system, to activate the expression of miRNAs within the DLK1-DIO3 genomic region and subsequently evaluate its effects on proliferation and metastasis of bladder cancer. Successful completion of these experiments is supposed to illustrate the functional role of c-Met and miRNAs in bladder tumorigenesis and provide novel strategies for the targeted therapy of bladder cancer.

膀胱癌是泌尿道最常见的恶性肿瘤，阐明膀胱癌发生的分子机制进而寻求肿瘤标记物和治疗靶点具有重要意义。我们在前期研究中发现在膀胱癌标本中c-Met显著上调，而DLK1-DIO3印记域的50余个miRNAs却是显著下调，进一步的研究提示它们之间或能相互调控。因此，本研究拟从表观遗传学和转录因子调控两大途径探索c-Met对于DLK1-DIO3印记域miRNAs簇表达的影响，以及拟利用生物信息学和荧光素酶双报告系统等手段探索DLK1-DIO3印记域中可靶向调控c-Met的miRNAs筛选和验证，从而能够阐明c-Met和DLK1-DIO3印记域miRNAs簇相互调控的具体机制。此外，我们将探索利用新型CRISPR-on技术全面上调DLK1-DIO3印记域抑癌miRNAs簇的表达来抑制膀胱癌生长和转移的可能性。本项目的成功实施一方面将对膀胱癌的发生机制作一补充，另一方面将为肿瘤的靶向治疗提供新的策略。

项目背景.膀胱癌是泌尿道最常见的恶性肿瘤，直接威胁着众多患者的生存健康和生活质量。在全世界范围内，它是男性中第七常见的肿瘤，而在女性中则排在第十位之后。..主要研究内容.1）DLK1-DIO3印记域中靶向调控c-Met的miRNAs筛选和验证。.2）c-Met对于DLK1-DIO3印记域miRNAs的调控。.3）CRISPR-on系统激活DLK1-DIO3印记域miRNAs簇靶向治疗膀胱癌。..重要结果和关键数据.本项目组在研究中发现DLK1-DIO3印记域miRNAs簇在膀胱癌中呈显著的一致低表达模式，基于临床样本和TCGA数据的预后因素分析提示DLK1-DIO3印记域miRNA簇的表达量与患者总生存时间呈显著有关。DLK1-DIO3印记域的miRNA簇的表达抑制与印记调控区基因间DMR（IG-DMR）甲基化有关。.上调DLK1-DIO3印记域miRNA的表达量，如miR-409-3p、miR-433、miR-323、miR-381和miR-300可以抑制膀胱癌细胞的增殖、细胞周期和迁移侵袭，并促进凋亡。c-Met信号通路介导了部分DLK1-DIO3印记域miRNA簇的下游调控，如c-MET/SMAD3/SNAIL轴介导了miR-323a-3p对于膀胱癌细胞生物学功能的调控，同时c-Met信号通路又可以负反馈调控DLK1-DIO3印记域miRNA簇的表达，从而形成一个闭合调控环路。.除了靶向癌基因c-Met以为，DLK1-DIO3印记域miRNAs簇的miR-300可以靶向SP1，miR-323a-3p可以靶向SMAD3，miR-433可以靶向CREB1，miR-381-3p可以靶向CDK6和CCNA2。这些研究结果进一步丰富了DLK1-DIO3印记域miRNA在膀胱癌中的调控网络。.我们设计了针对DLK1-DIO3印记域的sgRNA，并利用CRISPR-on系统成功在膀胱癌细胞中全面上调了DLK1-DIO3印记域的miRNAs簇的表达量，其发挥出了显著抑癌作用。..科学意义.本项目从c-Met和DLK1-DIO3印记域miRNAs簇之间的调控环路这一全新角度，系统性地探索膀胱癌发生和进展的机制，深化了对于非编码RNA和编码RNA之间调控网络的认识。此外，本项目在国内外首次探索利用CRISPR-on系统上调抑癌miRNAs来治疗恶性肿瘤的可能性，具有重要科学意义。