靶向启动子的hsa-miR-337抑制神经母细胞瘤中MMP-14表达的机制研究

Our previous studies indicated that matrix metalloproteinase-14 (MMP-14) promoted the invasion, metastasis and angiogenesis of neuroblastoma (NB). However, the underlying mechanisms for its expression and interference approaches still remain largely unknown. Transcription factor myeloid zinc finger 1 (MZF1) was able to drive the promoter activities of MMP-14 gene in NB, which was inhibited by endogenous microRNA-337 (hsa-miR-337) targeting its adjacent loci. Therefore, we hypothesized that hsa-miR-337 might inhibit the MMP-14 expression of NB cells through attenuating the binding of transcription factor MZF1 on its loci within MMP-14 promoter. On this basis, this project will investigate the regulatory functions of transcription factor MZF1 on MMP-14 expression in NB tissues and cells, define the effects and mechanisms of hsa-miR-337-mediated enrichment of AGO1/AGO2 and methylated histone in impacting the binding of MZF1 on MMP-14 promoter, and observe the roles of hsa-miR-337 in regulating MMP-14 expression, and the invasion, metastasis and angiogenesis of NB cells in vitro and in vivo. Successful accomplishment of this study will enrich the regulation mechanisms of MMP-14 expression from the insights of small non-coding RNA, and provide novel approaches and experimental basis for the treatment of NB.

我们前期发现：基质金属蛋白酶 14（MMP-14）促进神经母细胞瘤(NB)的侵袭、转移及血管生成，但其表达调控机制及干预途径不明；转录因子髓样锌指蛋白1（MZF1）能促进NB中MMP-14基因的启动子活性，且被靶向其调控位点邻近区域的内源性微小RNA 337（hsa-miR-337）所拮抗。因此我们假设：hsa-miR-337可能通过拮抗MZF1与MMP-14启动子的结合，抑制NB细胞中MMP-14的表达。本项目拟进一步解析MZF1对NB中MMP-14表达的调控作用，明确hsa-miR-337介导的AGO1/AGO2及甲基化修饰的组蛋白募集对MZF1结合至MMP-14启动子活性的影响及分子机制，观测hsa-miR-337抑制MMP-14表达对NB细胞体内外侵袭、转移及血管生成的影响，旨在从内源性小非编码RNA角度丰富MMP-14基因表达的调控机理，为NB的治疗提供新途径和实验基础。

基质金属蛋白酶14(MMP-14)促进神经母细胞瘤(NB)的侵袭、转移及血管生成，本课题通过建立MZF1过表达/敲低体系、hsa-miR-337过表达/拮抗以及AGO1/AGO2 敲低体系，解析神经母细胞瘤组织和细胞中hsa-miR-337、转录因子MZF1以及MMP-14基因表达的相关性，观测AGO1、AGO2、H3K9me2、H3K27me3在MMP-14启动子区域的富集及MZF1结合MMP-14启动子靶位点的变化情况，并在体内外研究hsa-miR-337调控MMP-14表达对神经母细胞瘤侵袭、转移及血管生成的影响，结果显示hsa-miR-337可以通过靶向MMP-14启动子从而调控MMP-14基因的启动子活性及基因转录，从而抑制神经母细胞瘤侵袭、转移及血管生成。在胃癌细胞中，MZF1能通过直接靶向MMP-14启动子从而上调MMP-14转录，miR-337-3p能通过募集AGO2下调MMP-14的转录。综合上述结果，本研究为探讨内源性小非编码 RNA 对MMP-14 基因表达的调控机理奠定了坚实的工作基础，为神经母细胞瘤的治疗提供新途径和实验基础，具有重要的理论指导意义和潜在的临床应用价值。项目资助发表SCI论文11篇。参与培养硕士生5名，其中1名已经取得博士学位，4名在读。项目投入经费23.0万元，支出21.4285万元，各项支出基本与预算相符。剩余经费1.57152万元，剩余经费计划用于本项目研究后续支出。