miR-221调控NLK/Wnt/N-myc信号促进神经母细胞瘤增殖的作用及机制研究

Amplification of N-myc oncogene is closely associated with the malignant phenotype and progression of neuroblastoma (NB). However, the mechanism of N-myc amplication is not fully clear. It has been reported that miR-221 was one of the marker molecules in the neuroblastoma with amplified N-myc.Our previous studies found that overexpression of miR-221 caused an enhancement of N-myc. Moreover, we indicated that NLK, one of the vital control factor of Wnt signaling pathway, was a target for miRNA-221. It is tempting to speculate that miRNA-221 activates Wnt pathway by targeting of NLK, and thus, induces transcriptional activation and persistent amplification of N-myc, and thereby may contribute to the progress of NB. .In this project, miR-221 was taken as the breakthrough point, and NB cell lines, SK-N-DZ and SH-SY5Y, in which N-myc is known to be overexpressed and poorly expressed respectively, were chosen as research objects. The effect of miRNA-221 on the Wnt signaling pathway by targeting of NLK will be discussed. Further, how the NLK/Wnt/N-myc signals affect the proliferation of NB cells and the progression of NB will be investigated. The present study may help to elucidate the mechanism of N-myc amplification in neuroblastoma.

神经母细胞瘤（NB）中N-myc扩增与其恶性表型和快速进展密切相关，然而促发N-myc扩增的机制尚未完全阐明。文献报道miR-221是N-myc扩增NB的标志miRNA，我们前期工作证实上调miR-221表达，可引起N-myc表达增加，而且发现Wnt信号途径的重要调控因子NLK是miR-221的靶基因。因此提出"miR-221通过靶向抑制NLK，参与Wnt信号调控，促发N-Myc的转录激活和持续扩增，导致NB恶性增殖"的假设。.本课题以miR-221为切入点，以NB细胞株SK-N-DZ（N-myc扩增）和SH-SY5Y（N-myc无扩增）为研究对象，通过体内外实验，分别上调和下调NB细胞中miR-221的表达，研究其靶向抑制NLK对Wnt信号途径的调控作用，以及NLK/Wnt/N-myc信号如何影响NB细胞增殖及恶性进展。本研究有助于阐明NB中N-myc扩增的机制。

神经母细胞瘤（NB）中N-myc扩增与其恶性表型和快速进展密切相关，然而促发N-myc扩增的机制尚未完全阐明。.本课题以miR-221(文献报道miR-221是N-myc扩增NB的标志miRNA)为切入点，以NB细胞株IMR-32（N-myc扩增/高表达）和SH-SY5Y（N-myc无扩增/正常表达）为研究对象，通过体内外实验，分别上调和下调NB细胞中miR-221的表达，研究其靶向抑制NLK对Wnt信号途径的调控作用，以及NLK/Wnt/N-myc信号如何影响NB细胞增殖及恶性进展。.我们的研究结果发现：(1)NB患儿的肿瘤组织和NB细胞系中N-myc表达与miR-221呈正相关；NB患儿中miR-221的高表达与其预后密切相关；上调NB细胞中miR-221表达，可引起N-myc表达增加；(2)通过生物信息学和实验手段证实Wnt信号途径的重要调控因子NLK是miR-221的靶基因；(3)通过体外实验，分别上下调miR-221，发现miR-221可以抑制NLK表达，降低LEF1磷酸化水平，上调N-myc表达；体内实验亦证实上调miR-221可导致NLK表达降低，p-LEF1表达降低，而N-myc表达增加；(4)体外实验发现，利用siRNA干扰技术，敲低NLK的表达，可降低p-LEF1表达，并上调N-myc的表达；上调NLK的表达后，导致p-LEF1表达增加，同时N-myc的表达降低；(5)体外实验发现，miR-221可通过抑制p21、p27、p57，促进细胞通过G1/S期阻滞；体内外实验均表明miR-221可促进细胞增殖。.上述研究结果表明，miR-221在N-myc高表达的NB肿瘤组织和NB细胞系中表达明显增加，并且与NB患儿的预后不良密切相关；miR-221通过靶向抑制NLK，降低LEF-1磷酸化水平，参与Wnt信号调控，促发N-Myc的表达增加；miR-221可通过促进细胞通过G1/S细胞周期关卡，促进细胞增殖；此外，我们亦发现miR-221可抑制细胞周期细胞因子p21，基于文献报道N-myc可通过抑制p21促进细胞周期进展，而现有的生物信息学和实验证据并不支持p21是miR-221的靶基因，因此，miR-221对促进细胞增殖的作用至少部分是通过靶向调控NLK所致的N-myc蛋白表达上调实现的，进而促进细胞通过G1/S细胞周期关卡，导致NB细胞增殖和恶性进展。

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