BACKGROUND The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

中文翻译：

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PLAGL2 / MYCN / miR-506-3p相互作用调节神经母细胞瘤细胞的命运并与神经母细胞瘤的进展相关。

背景技术癌基因MYCN对于包括神经母细胞瘤在内的几种类型的癌症的肿瘤发生至关重要。我们先前曾报道miR-506-3p抑制了神经母细胞瘤细胞中的MYCN表达。然而，由于MYCN 3'UTR中没有miR-506-3p靶位，因此尚不清楚这种调控的机制。方法通过结合芯片，信息学和荧光素酶报告基因检测的系统研究，我们确定转录因子多态性腺瘤样基因2（PLAGL2）是miR-506-3p的直接靶标，介导其对MYCN表达的调控。使用CHIP-PCR和荧​​光素酶报告基因分析，我们验证了PLAGL2对MYCN的转录调控，并进一步证明了MYCN对PLAGL2的转录调控。我们通过细胞活力测定，集落形成和分化标记物的Western印迹检查了PLAGL2在调节神经母细胞瘤细胞命运中的功能。我们通过定量PCR和Western印迹检查了视神经母细胞瘤治疗中使用的分化剂视黄酸对miR-506-3p，PLAGL2和MYCN表达的影响。我们通过使用公共神经母细胞瘤患者数据集检查肿瘤PLAGL2 mRNA水平与MYCN mRNA表达和患者生存率的相关性，研究了PLAGL2表达的临床相关性。结果我们发现miR-506-3p直接下调了PLAGL2的表达，并验证了MYCN启动子区域中负责促进MYCN转录的PLAGL2结合位点，从而建立了miR-506-3p调节MYCN表达的机制。反过来，我们发现MYCN通过PLAGL2启动子区域中的五个N-Myc结合E-boxs调节PLAGL2转录。我们进一步证实了多种神经母细胞瘤细胞系中内源性PLAGL2和MYCN之间的相互调节。此外，我们发现PLAGL2敲低诱导神经母细胞瘤细胞分化并减少细胞增殖，并且PLAGL2和MYCN的组合敲低显示协同作用。更令人惊讶的是，我们发现在神经母细胞瘤患者中，高肿瘤PLAGL2 mRNA水平与高MYCN mRNA水平和不良患者生存率显着相关。此外，我们发现视黄酸增加了miR-506-3p的表达，并抑制了MYCN和PLAGL2的表达。结论我们的研究结果完全表明，由PLAGL2形成的相互作用网络