NKX2.1双重调控TrkA和TrkB的表达抑制神经母细胞瘤生长的作用与机制研究

Neuroblastoma, the most common extracranial malignancy in children, is featured with dramatic heterogeneity in both the clinical behavior and genetic characteristics. Some patients have a high likelihood of undergoing spontaneous regression to benign tumors without any interventional therapy. However, the underlying mechanism is currently unknown. Mounting evidence shows that expression levels of TrkA and TrkB are able to control cell proliferation, apoptosis and differentiation of neuroblastoma, and represent important biomarkers for determining cytogenetic subtypes of neuroblastoma. Nevertheless, the mechanism underlying regulation of TrkA and TrkB expression is far from clearly understood. Recently, our preliminary results demonstrated that the transcriptional factor NKX2.1 was significantly increased in more differentiated neuroblastoma tissues. In neuroblastoma cell lines, NKX2.1 not only increased TrkA expression at both the mRNA and protein levels, but also decreased TrkB rotein expression, as well as unregulated transcription of miR-204. Notably, TrkB was predicted to be a putative potential target of miR-204, which was shown to downregulate TrkB expression in neuroblastoma cell lines. Therefore, in the current project, we are aiming to further investigate the inhibitory effect of NKX2.1-induced dual regulation of TrkA and TrkB in growth of neuroblastoma cells and contribution of proliferation, apoptosis, and differentiation to NKX2.1-mediated growth inhibition, which would help to clarify molecular mechanisms underlying heterogeneity of neuroblastoma, and provide novel molecularly diagnostic and prognostic target for neuroblastoma patients.

神经母细胞瘤（NB）是儿童最常见的颅外恶性实体肿瘤， 其临床表现和生物学特性具有显著异质性，部分NB可向良性分化甚至完全消退，但是其分子机制尚不清楚。研究表明，TrkA和TrkB基因的表达控制NB细胞增殖、凋亡和分化，是划分NB基因亚型的重要标志物，但其调控机制尚不清楚。我们前期通过芯片筛选发现转录因子NKX2.1的表达随着NB的分化程度升高而明显增加。在NB细胞中，NKX2.1既上调TrkA的蛋白和mRNA表达，也能下调TrkB的蛋白表达，并上调miR-204的转录水平，而miR-204可减少TrkB的表达。生物信息学预测发现TrkB是miR-204的可能潜在靶蛋白。功能上，外源性NKX2.1显著抑制NB细胞的体外生长。在此基础上，本项目将深入阐明NKX2.1双重调控TrkA和TrkB表达从而抑制NB细胞生长的生物学作用与分子机制，从而有助于阐明NB异质性，为NB的诊断和预后提供新靶点。

TrkA和TrkB基因的表达控制NB细胞增殖、凋亡和分化，但其调控机制尚不清楚。我们发现了下列结果。.1. 以GN为对照，高分化NB和未分化NB均呈现出显著不同的基因表达。信号通路分析提示：TTF1在GN组织中表达上调，但在未分化的NB组织中表达下调；TrkA在GN组织及高分化的NB组织中表达上调；但在未分化的NB组织中表达下调；TrkB在GN、高分化NB和未分化NB中均表达较低。25株NB细胞系TTF1和TrkA的mRNA显著低于4株视网膜色素上皮细胞系；TrkB的mRNA在NB细胞系及视网膜色素上皮细胞系中呈均一表达。.2. TTF1和TrkA在GN表达较高，高分化NB次之，未分化NB组织中表达最低。但是，TrkB在未分化NB表达最高，高分化NB次之、GN最低。进一步免疫组化显示TTF1和TrkA在GN中表达较高、高分化NB中次之、未分化NB中表达最低；TrkB在未分化NB中表达较高、高分化NB中次之、GN中表达最低。TTF1的表达与TrkA呈正相关、与TrkB呈负相关。.3. 通过焦磷酸测序分析和甲基化特异PCR，我们发现：相较于GN，未分化NB中TTF1甲基化显著高于GN，高分化NB次之。TTF1甲基化导致TTF1在未分化NB中表达下降。此外，我们确定了SK-N-BE及SK-N-SH细胞系中TTF1的甲基化状态。结果显示SK-N-BE细胞系中TTF1甲基化显著高于SK-N-SH细胞系。.4.在SK-N-BE细胞系中过表达TTF1可显著提高TrkA的mRNA水平。在SK-N-BE细胞系中过表达TTF1对TrkB的mRNA无显著影响。此外，过表达TTF1可显著提高SK-N_BE细胞中TrkA蛋白的表达。但是过表达TTF1导致SK-N-BE中TrkB蛋白的表达下降。软琼脂实验显示过表达TTF1显著抑制肿瘤细胞集落形成能力。EdU分析显示过表达TTF1显著抑制细胞增殖。我们采用两个独立的siRNAs敲减TTF1在SK-N-SH细胞系中的表达。结果显示敲低TTF1导致TrkA表达降低，但是敲低TTF1却恢复了TkrB的表达。同时，沉默TTF1，导致SK-N-SH细胞系集落形成能力显著增强。此外，沉默TTF1导致SK-N-SH细胞系肿瘤增殖能力显著增强。.综上所述，NKX2.1双重调控TrkA和TrkB表达从而抑制NB细胞生。