Evaluation of JARID1B expression and stemness functions in <i>MYCN</i> and <i>non-MYCN</i> neuroblastoma (NB) cell lines.

<p>(A) Western blot analysis was performed to investigate the expression of JARID1B and α-MYCN in <i>MYCN</i> amplification (MNA⁺) and <i>non-MYCN</i> amplification (MNA^-) NB cells. (B) The SP percentage was analyzed in MNA⁺ and MNA^- NB cells by Hoechst staining and flow cytometry. SK-N-BE(2) and SK-N-AS cells had 9.05% and 1.45% SP cells (C) Representative Aldeflour assay result of MNA⁺ SK-N-BE(2) and MNA^- SK-N-AS NB cells showed 17.2% and 3.43% ALDH+ subpopulation, respectively. Data was collected from three independent experiments.</p

Silencing JARID1B decreases epithelial to mesenchymal transition (EMT).

<p>(A) Western blot analysis of epithelial and mesenchymal markers’ expression in wild type and JARID1B-silenced SK-N-BE(2) cells show downregulation of N-cadherin and vimentin in response to JARID1B knockdown, while E-cadherin was upregulated. β-actin served as loading control. (B) Bar chart quantification of (A). Expression of EMT markers are normalized against β-actin. Assay was performed three times. *, represent p<0.05.</p

JARID1B-silencing suppresses tumorsphere formation and increases cisplatin sensitivity.

<p>(A) Tumorspheres generated from wild type and JARID1B-shRNA 1- and 2- infected SK-N-BE(2) cells. (B and C) JARID1B knockdown caused significant reduction in number and sizes of tumorspheres formed. (D) Cell viability assay show that the cytotoxicity effect of cisplatin was significantly enhanced by JARID1B silencing. All assays were repeated at least three times. * and ** indicate p<0.05 and p<0.001.</p

JARID1B downregulation results in the decrease of cell invasion capability.

<p>(A) Western blots confirmed JARID1B expression was suppressed in the two shRNA clones 1 and 2 as compared to the control, and there was correlative down-regulation of Nestin and BCL-xL, while BAX was up-regulated. (B) Wound healing migration assay of SK-N-BE(2) cells show that JARID1B-shRNA infected cells were less motile than control wild type cells (C) Matrigel invasion assay and histogram representation show that JARID1B knockdown induced very significant downregulation of the invasive potential of the SK-N-BE(2) cells (**, p<0.01).</p

Silencing JARID1B downregulates Jagged/Notch signaling transduction pathway.

<p>The expression of Notch and its ligand in wild type and JARID1B-silenced SK-N-BE(2) cells were analyzed by Western blot assay. (A) JARID1B-silencing disrupted Jagged1, Notch1 and Notch 2 signaling. (B) Comparative bar diagrams demonstrate the downregulation of Notch signaling in the wild type SK-N-BE(2) cells, compared to the JARID1B-silenced SK-N-BE(2) cells. The intensity was measured relative to loading control (β-actin) from three independent experiments. *, p<0.05 (C) Immunofluorescent staining showing that JARID1B-silencing downregulated the expression of Notch1. Notch1 (green) colocalizes with JARID1B (red) in the nucleus (DAPI for nuclear staining, blue).</p

Aryl Hydrocarbon Receptor Downregulates MYCN Expression and Promotes Cell Differentiation of Neuroblastoma

<div><p>Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. <i>In vitro</i> studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.</p></div

Neuroblastoma SP cells with higher JARID1B expression are more resistant to chemotherapeutics.

<p>(A) The morphology of SK-N-BE(2), SK-N-DZ, SK-N-AS and SK-N-SH spheroid. (B) Comparative analysis of JARID1B expression in SK-N-BE(2) and SK-N-AS cells, as well as the tumorsphere derived from either cells, SK-N-BE(2)-S and SK-N-AS-S show that the tumorspheres were more enriched in JARID1B and Nestin protein. (C) Immunofluorescence analysis shows SK-N-BE(2) and SK-N-AS tumorspheres expressing JARID1B (red) and Nestin (green) and nucleus marker, DAPI, blue. (D) SRB assay showing that the tumorspheres SK-N-BE (2)-S were more resistant to chemotherapeutics than the parental SK-N-BE (2) cells. Error bars represent the SD from three independent experiments. ** p < 0.01.</p

The first rank of significant genetic networks in differentially expressed genes in neuroblastoma.

<p>The first rank of significant genetic networks in differentially expressed genes in neuroblastoma.</p

AHR regulates MYCN expression level in NB cell lines.

<p>(A) SK-N-SH cells were transfected with increasing amounts of AHR by a lentivirus transduction system. The cell lysate was collected by lysis buffer with proteinase inhibitors and analyzed by Western Blot. (B) The SK-N-DZ cell line was transfected with human AHR expression vector (pEGFP-C1-hAHR) by Lipofectamine 2000 and selected by G418 antibiotics. By morphology observation, AHR overexpression promotes neurite outgrowth of SK-N-DZ cell line. (C) 1 µg of total RNA, isolated by TRIzol reagent from SK-N-DZ/AHR cells, was reverse-transcribed to cDNA. The expression levels of each gene were quantified by SYBR-Green real-time PCR. AHR overexpression in SK-N-DZ cells suppressed E2F1 and MYCN expression and upregulated the mRNA expression level of differentiation markers, GAP43 and CRT. (D) Tet21N is a neuroblastoma cell line with tetracycline-controlled expression of MYCN (Tet-off). 10 ng/ml tetracycline was added to the growth media for 24 h to conditionally knockdown MYCN expression. After tetracycline treatment, the total mRNA were collected and subjected to real-time PCR analysis with gene specific primers. (E) SK-N-SH cells were transfected with AHR shRNA by lentivirus transduction system. After 48 h, total mRNA were collected and subjected to real-time PCR with specific primers for AHR, E2F1, MYCN, Nestin, and Vimentin.</p

The correlation between AHR and MYCN mRNA expression level and differentiation histology in tumor samples of patients with NB.

<p>(A) Total mRNA of patient tumor samples were isolated by TRIzol reagent. The mRNA expression level of AHR and MYCN were analyzed using SYBR-green real-time PCR using gene specific primers. (B) The inverse correlation were analyzed by Spearman’s correlation test (Spearman’s ρ = −0.6484, p = 0.0121) (C) AHR expression level was analyzed in tumors with high and low MYCN expression status. (D) The total mRNA of MYCN non-amplified NB cell lines, SY5Y and SK-N-SH, and MYCN amplified NB cell lines, SK-N-BE, SK-N-DZ, and IMR32, were collected and subjected to SYBR-green real-time PCR with specific primers for AHR and MYCN. (E) The correlation between AHR expression level and differentiation of NB tumor histology was analyzed in 34 human NB tumor samples.</p