The PLAGL2/MYCN/miR-506-3p interplay regulates neuroblastoma cell fate and associates with neuroblastoma progression

Abstract

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.

Long non-coding RNA MAPKAPK5-AS1/PLAGL2/HIF-1α signaling loop promotes hepatocellular carcinoma progression

“…Previous studies have reported that pleomorphic adenoma gene like-2 (PLAGL2), as a member of the PLAG zinc finger transcription factor, is widely involved in the progress of various tumors [ 40 – 42 ], including HCC. For example, Liang Wu et al confirmed that PLAGL2 promoted epithelial-mesenchymal transition and mediated colorectal cancer metastasis via β-catenin-dependent regulation of ZEB1 [ 42 ].…”

Molecular Mechanisms of MYCN Dysregulation in Cancers

“…Other transcription factors, such as specific protein 1 (SP1) ( 88 ), E2F ( 89 ), and pleiomorphic adenoma gene-like 2 (PLAGL2) ( 90 ), participate in the regulation of MYCN expression. The three transcription factors directly bind to the cognate binding sites in the MYCN promoter, contributing to MYCN activation.…”

“…The three transcription factors directly bind to the cognate binding sites in the MYCN promoter, contributing to MYCN activation. Moreover, N-MYC regulates PLAGL2 transcription through five N-MYC-binding E-boxes in the PLAGL2 promoter region, forming a positively regulatory loop between the two transcription factors, which is crucial for expression of each other in neuroblastoma tumors ( 90 ). Lipid desaturation-associated endoplasmic reticulum (ER) stress inhibits MYCN expression via upregulating the transcriptional repressor ATF3 in hepatocellular carcinoma cells ( 91 ).…”

“…All-trans retinoic acids have been used for neuroblastoma therapy for decades by inhibiting the expression of MYCN and inducing the neuronal differentiation of neuroblastoma cells ( 94 – 96 ). Loss of E2F binding or suppression of PLAGL2 expression mediates the negative regulation of MYCN expression by retinoic acid ( 89 , 90 ). Acyclic retinoid dampens MYCN gene expression and suppresses cell proliferation of MYCN -overexpressed hepatocellular carcinoma cells, at least in part by ER stress-induced ATF3 signaling pathway ( 91 ).…”

“…Along with transcription factors, noncoding RNAs including long noncoding RNA (lncRNAs) and microRNAs (miRNAs) are involved in the regulatory network of MYCN expression. miR-506-3p is a potent differentiation inducer and a strong repressor of MYCN expression in neuroblastoma cells by targeting PLAGL2 transcription factor ( 90 , 104 ). miR-204 directly binds MYCN mRNA, represses MYCN expression, and inhibits a subnetwork of oncogenes that strongly correlate with MYCN -amplified neuroblastoma and poor patient outcome ( 105 ).…”