CTCF Cooperates With Long Non-coding RNA MYCNOS To Facilitate MYCN Expression In Neuroblastoma And Its Underlying Mechanisms

Part I Transcription factor CTCF facilitates the expression of MYCN in NB cellsObjective:To search for the crucial transcription factors that regulate the expression of MYCN in neuroblastoma, and to evaluate its effects on MYCN gene promoter activity and gene expression in neuroblastoma.Methods:Overlapping analysis of publicly available NB microarray data in R2 database, UCSC Genome Bioinformatics Browser, TF binding sites analysis software Genomatix Matlnspector, ChlPBase and ChlP-X database was performed to search for the crucial transcription factors that regulate the expression of MYCN in neuroblastoma and localize their binding sites within MYCN promoter. Dual-luciferase assay was utilized to evaluate their effects on MYCN promoter activity. qRT-PCR and western blot assays were performed to detect their effects on the transcript and protein levels of MYCN.Results:Comprehensive analysis of multiple databases and platforms revealed CTCF as the most important transcription factor that regulate the promoter activity and expression of MYCN gene. The results of dual-luciferase, qRT-PCR and western blot assays indicated that ectopic expression or knockdown of CTCF increased and decreased the MYCN promoter activity, respectively, and mutation of two CTCF binding sites within MYCN promoter abolished these effects. qRT-PCR and western blot assays further demonstrated that ectopic expression or knockdown of CTCF obviously increased and decreased the transcript and protein levels of MYCN, respectively.Conclusions:Transcription factor CTCF targets the binding sites within MYCN promoter, and activates the MYCN gene promoter activity, resulting in up-regulation of MYCN expression in neuroblastoma.Part ? Nature antisense lncRNA MYCNOS promotes the expression of MYCN in NB cellsObjective:To elucidate the most important MYCNOS isoform in neuroblastoma, and to evaluate its effects on MYCN gene promoter activity and gene expression in neuroblastoma.Methods:RT-PCR was performed to examine the expression profiles of 5 MYCNOS isoforms in NB tissues and cell lines. The EGFP fusion vector for the potential open reading frame (ORF) of MYCNOS was constructed to investigate the protein-coding potential of MYCNOS. Dual-luciferase assay was utilized to evaluate its effects on MYCN promoter activity. qRT-PCR and western blot assays were performed to detect its effects on MYCN transcript and protein levels.Results:RT-PCR revealed that the 770 bp-length isoform was the mainly expressed MYCNOS in NB tissues and cell lines. Ribosome profiling data shows low protein-coding potential of MYCNOS. Transfection of EGFP fusion vector for the potential ORF of MYCNOS resulted in no obvious expression of MYCNOS protein in NB cells as revealed by immunofluorescence and western blot detection. Dual-luciferase, qRT-PCR and western blot assays demonstrated that ectopic expression or knockdown of MYCNOS increased and decreased promoter activity and transcript and protein levels of MYCN in NB cells, respectively, and these effects were not affected by point mutation that abolished the protein-coding potential of MYCNOS.Conclusions:The 770 bp-length isoform is the mainly expressed MYCNOS in NB tissues and cell lines. MYCNOS-770 bp promotes the transcription and expression of MYCN in neuroblastoma, which is independent of its protein-coding potential.Part III Functional interaction between CTCF and MYCNOS in regulating the MYCN expression in NB cellsObjective:To explore the physical interaction between CTCF protein and MYCNOS RNA, and to investigate the cooperative effects of CTCF with MYCNOS on the transcription and expression of MYCN in neuroblastoma.Methods:The catRAPID algorithm was used to estimate the binding propensity of CTCF protein and MYCNOS RNA. RIP, RNA pull-down, RNA-EMSA and in vitro binding assays were performed to investigate the actual interaction between CTCF and MYCNOS. Dual-luciferase, qRT-PCR and western blot assays were performed to evaluate the cooperative effects of CTCF with MYCNOS on MYCN gene promoter activity and gene expression in neuroblastoma.Results:The catRAPID algorithm analysis revealed a high interaction propensity between CTCF protein and MYCNOS RNA. RIP and RNA pull-down assays detected the physical interaction between CTCF and MYCNOS. RIP and RNA-EMSA assays indicated that exon 1 and exon 3 of MYCNOS interacted with CTCF protein with great potential. In vitro binding assay using recombinant CTCF indicated that the zinc-finger domain of CTCF was crucial for the interaction with MYCNOS. Dual-luciferase assay indicated that knockdown of CTCF or MYCNOS abolished the enhanced MYCN promoter activity induced by stable transfection of MYCNOS or CTCF, respectively. RIP, qRT-PCR and western blot assays indicated that knockdown of CTCF or MYCNOS abolished their interaction, and attenuated the expression of MYCN induced by MYCNOS or CTCF, respectively.Conclusions:Transcription factor CTCF physically interacts with MYCNOS, and then functions cooperatively to promote the transcription and expression of MYCN in neuroblastoma.Part IV MYCNOS facilitates the recruitment of CTCF and chromatin remodeling at MYCN promoter in NB cellsObjective:To explore the mechanisms underlying the cooperative functions of CTCF with MYCNOS on the gene activation and up-regulation of MYCN in neuroblastoma.Methods:ChIP assay was performed to detect the binding of CTCF on MYCN promoter and evaluate the influence of MYCNOS on CTCF enrichment level. NB cells were treated with DNA methyltransferase inhibitor to investigate whether DNA methylation status could affect the binding of CTCF on MYCN promoter and whether the transcription activation of MYCN gene by CTCF is induced through DNA demethylation. ChIP assay were performed to detect the influence of CTCF and/or MYCNOS on the chromatin status of MYCN promoter.Results:ChIP assay detected the binding of CTCF on MYCN promoter in NB cells, which was not affected by DNA methylation status. Knockdown or over-expression of MYCNOS decreased and increased the binding of CTCF to MYCN promoter respectively. Administration of DNA methyltransferase inhibitor resulted in no significant changes in MYCN transcript levels induced by knockdown of CTCF. Ectopic expression of CTCF resulted in increased binding of active markers of Histone 3 lysine 4 dimethylation (H3K4me2), Histone 3 lysine 4 trimethylation (H3K4me3) and RNA Polymerase II to MYCN promoter, and decreased the recruitment of repressive markers of Histone 3 lysine 9 trimethylation (H3K9me3) and Histone 3 lysine 27 trimethylation (H3K27me3), which was attenuated by knockdown of MYCNOS, and vice versa.Conclusions:Binding of transcription factor CTCF on MYCN promoter increases the recruitment of chromatin active markers and decreases the recruitment of chromatin repressive markers on MYCN promoter in NB cell lines, and MYCNOS facilitates the binding of CTCF on MYCN promoter.Part V CTCF cooperates with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivoObjective:To evaluate the effects of the cooperation function of CTCF and MYCNOS on the proliferation, invasion, metastasis and differentiation of neuroblastoma through regulating MYCN gene promoter activity and gene expression in vitro and in vivo.Methods:Subclone cell lines of neuroblastoma with CTCF stably transfected or knockdown and/or MYCNOS stably transfected or knockdown were established. The changes in cell proliferation and invasion capabilities were determined by soft agar assay, flow cytometry and matrigel invasion assay. The changes of neuronal differentiation markers were detected by immunofluorescence assays. In addition, the effects of CTCF and MYCNOS on the proliferation, invasion and metastasis of NB cells in vivo were evaluated in different nude mice models.Results:Over-expression of MYCNOS suppressed the differentiation and promoted the proliferation and invasion of NB cells in vitro, which could be antagonized by knockdown of CTCF. Over-expression of CTCF suppressed the differentiation and promoted the proliferation and invasion of NB cells in vitro, which could be antagonized by knockdown of MYCNOS. Over-expression of CTCF in NB cells significantly increased the tumor volume, tumor weight and the counts of lung metastases in nude mice, which could be antagonized by knockdown of MYCNOS.Conclusions:CTCF cooperates with MYCNOS to suppress the differentiation and promote the growth, invasion and metastasis of NB cells in vitro and in vivo.Part VI CTCF and MYCNOS are highly expressed and positively correlated with MYCN expression in NB tissues and cell linesObjective:To analyze the expression profile of CTCF and MYCNOS in different NB tissues and cell lines, and to evaluate their correlation with MYCN expression, tumor severity and prognosis of NB patients.Methods:qRT-PCR and western blot assays were performed to evaluate the expression of CTCF and MYCNOS in different NB tissues and cell lines. The NB data in R2 database were used to analysis the correlation of CTCF or MYCNOS expression with MYCN expression, tumor progression and prognosis of NB patients.Results:Immunohistochemical staining of 42 NB tissues revealed that CTCF expression was higher in cases with poor differentiation, higher mitosis karyorrhexis index, advanced International Neuroblastoma Staging System (INSS) stages or MYCN amplification, and the MYCN immunoreactivity was consistent with the immunoreactivity of CTCF. Mining the data in 88 NB cases on R2 database indicated that higher transcript levels of CTCF or MYCNOS were observed in NB cases with advanced INSS stages, death, MYCN amplification or recurrence/progression. The MYCN transcript levels were positively correlated with those of CTCF or MYCNOS in 30 NB tissues, consistent with the results from R2 database. Kaplan-Meier survival plots of 88 and 498 well-defined NB cases derived from R2 database revealed that patients with high CTCF or MYCNOS expression had lower survival probability than those with low expression. Cox regression analysis of the 498 NB cases indicated CTCF expression were independent prognostic factors for unfavorable outcome of NB patients.Conclusions:The expression of CTCF or MYCNOS is positively correlated with the expression of MYCN in NB tissues and cell lines, and high expression of CTCF or MYCNOS is correlated to tumor progression and poor prognosis of NB patients.