Department of Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX, 78666, USA.
J Exp Clin Cancer Res. 2020 Feb 22;39(1):41. doi: 10.1186/s13046-020-1531-2.
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.
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.
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.
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.
癌基因 MYCN 对包括神经母细胞瘤在内的几种癌症的肿瘤发生至关重要。我们之前报道过，miR-506-3p 可抑制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 的靶位点，因此这种调节的机制尚不清楚。
通过结合微阵列、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形性腺瘤基因样 2（PLAGL2）是 miR-506-3p 的直接靶标，介导其对 MYCN 表达的调节。通过 CHIP-PCR 和荧光素酶报告基因检测，我们验证了 MYCN 受 PLAGL2 的转录调控，并且进一步证明了 PLAGL2 受 MYCN 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 印迹分析，研究了 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 blot 检测了分化剂视黄酸对 miR-506-3p、PLAGL2 和 MYCN 表达的影响。我们通过检查公共神经母细胞瘤患者数据集，用肿瘤 PLAGL2 mRNA 水平与 MYCN mRNA 表达和患者生存的相关性，研究了 PLAGL2 表达的临床相关性。
我们发现 miR-506-3p 直接下调 PLAGL2 表达，并在 MYCN 启动子区域验证了一个 PLAGL2 结合位点，该位点负责促进 MYCN 转录，从而建立了 miR-506-3p 调节 MYCN 表达的机制。相反，我们发现 MYCN 通过 PLAGL2 启动子区域中的五个 N-Myc 结合 E 盒调节 PLAGL2 转录。我们进一步在多个神经母细胞瘤细胞系中证实了内源性 PLAGL2 和 MYCN 之间的这种相互调节。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 的联合敲低表现出协同效应。更引人注目的是，我们发现高肿瘤 PLAGL2 mRNA 水平与神经母细胞瘤患者中高 MYCN mRNA 水平和不良预后显著相关。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。
我们的研究结果表明，PLAGL2、MYCN 和 miR-506-3p 形成的相互作用网络是调节神经母细胞瘤细胞命运、决定神经母细胞瘤预后以及介导视黄酸治疗作用的重要机制。
