LncRNA AC142119.1 facilitates the progression of neuroblastoma by epigenetically initiating the transcription of MYCN
Journal of Translational Medicine volume 21, Article number: 659 (2023)
Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear.
Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter.
AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression.
We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.
Neuroblastoma (NB) is the most prevalent extra-cranial solid tumor in young children, which is a malignancy of the sympathetic nervous system, arising from the neural crest during the embryonic development [1, 2]. It accounts for about 6–10% of all childhood tumors but 12–15% of all cancer deaths in children . NB has been characterized with a high heterogeneity in clinical presentations, ranging from spontaneous regression tumors without any medical intervention to highly aggressive and metastatic diseases that are resistant to multimodal anti-cancer therapies [4, 5]. This heterogeneity leads to great differences in the survival of NB patients. The survival rate of patients diagnosed with low- and intermediate‐risk NB is greater than 90%, whereas the 5-year survival rate of the patients with high-risk NB is less than 50% [6, 7]. Due to the insidious symptoms, the majority of patients were diagnosed with high-risk NB, it is necessary to identify novel therapeutic targets to improve the prognosis of NB patients.
MYCN belongs to the MYC proto-oncogene family and encodes a transcription factor that modulates the expression of numerous target genes, which engage in a broad spectrum of intracellular biological process including cell proliferation, apoptosis, senescence, differentiation, metabolism, DNA damage repair and protein synthesis [8, 9]. Amplification of the MYCN gene has been one of the earliest genetic markers in NB and occurs in approximately 22% of all NB and 45% of high-risk NB cases [10, 11]. MYCN gene amplification and consequent MYCN oncoprotein overexpression are strongly correlated with particularly poor prognosis of NB patients, and defines an aggressive subset of NB . Laboratory evidences also indicated that exogenous expression of MYCN in NB cells with non-amplified MYCN enhanced the cell proliferation and tumorigenic capacity . Additional studies further suggested that ectopic expression of MYCN is sufficient to drive MYCN-amplified NB in mice and zebrafish models [13, 14]. However, the underlying mechanisms of MYCN overexpression in NB are not fully illustrated up to date.
Long non-coding RNAs (lncRNAs) are a class of transcripts with a length of more than 200 nucleotides that lack the ability to encode proteins . Recent years, lncRNAs have been proposed to serve as key regulators encompassed complex functions in various human cancers, including NB, and have attracted increasing attention . Previous studies demonstrated that lncRNAs could regulate epigenetic modification, gene transcription, translation, the stability of RNA/protein and post-translational modification via interacting with DNA, RNAs and/or proteins, thus affecting the biological behaviors of cancer cells [17,18,19]. In NB, it has been demonstrated that lncRNA HNF4A-AS1 enhanced the aerobic glycolysis and growth of NB cells by transactivation of CTCF and transcriptional alteration of HNF4A via binding with hnRNPU . Moreover, lncRNA NHEG1 physically interacted with and stabilized DDX5 protein, resulting in the transactivation of β-catenin and progression of NB . However, only a few such lncRNAs have been well-characterized in NB, the biological function and underlying mechanisms of the great majority of these transcripts in the initiation and progression of NB remain unexplored.
Here, we identified a novel MYCN-related lncRNA AC142119.1, which was significantly upregulated in MYCN-amplified NB tissues and cell lines, compared with those without MYCN amplification, and high expression of AC142119.1 was positively associated with poor survival in NB patients. Furthermore, we uncovered that AC142119.1 could recruit WDR5 protein to interact with the promoter of MYCN, further activate the transcription of MYCN and eventually accelerate the progression of NB. The data present here provided novel insights into the potential mechanism by which AC142119.1 contributed to the overexpression of MYCN in NB, implying that AC142119.1 might be a promising therapeutic target for NB.
Human neuroblastoma cell lines (SH-SY5Y, SK-N-AS, IMR-32 and SK-N-DZ) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All these cells were maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37 ℃ with 5% CO2 in a humidified atmosphere.
The 55 cases of NB tissue specimens were collected from the randomly selected patients who were diagnosed with NB at Children’s Hospital of Chongqing Medical University (Chongqing, China) in compliance with routine clinical sampling after obtaining the written informed consent from their patient or legal guardian. None of these patients received chemotherapy or radiotherapy prior to surgical resection. The present study conformed to the Declaration of Helsinki and was approved by the Ethics Committee of Children’s Hospital of Chongqing Medical University (Chongqing, China).
RNA isolation, microarray analysis
Total RNA of NB cells and tissues were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The purity and quantity of the isolated RNA were evaluated by Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA integrity was examined by denaturing agarose gel electrophoresis. The Arraystar Human LncRNA microarray V3.0 (Arraystar, USA) was applied to obtain the lncRNA expression profile. The sample labeling, microarray hybridization and data analysis were conducted by Kangcheng Bio-tech (Shanghai, China).
Rapid amplification of cDNA ends (RACE) analysis
RACE assay was performed with GeneRacer™ Kit (Invitrogen) in accordance with the manufacturer’s instructions to determine the full length of AC142119.1 transcript in SK-N-DZ cells. The products of RACE PCR were resolved on 1.5% agarose gel. The gel products were purified using SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) and cloned into pGM-T vector for sequencing. The specific primers were listed in Additional file 1: Table S1.
Total RNA was reverse transcribed into cDNA with PrimeScript RT-PCR Kit (Takara, Dalian, China) based on the manufacturer’s protocols. Then the cDNA was amplified with SYBR Green Premix Ex Taq (Takara). The relative expression of the target genes was assessed by 2–ΔΔCT method, with GAPDH serving as an internal reference. The primer sequences were displayed in Additional file 1: Table S1.
Vectors, GapmeRs, siRNAs and cell transfection
The full-length cDNA of AC142119.1 was synthesized and inserted into the lentiviral vector pHBLV-CMVIE-ZsGreen-T2A-puro to establish AC142119.1 overexpression vector, with the empty vector without AC142119.1 sequence serving as a control. To knockdown AC142119.1, LNA-modified antisense oligonucleotides (GapmeRs) targeting AC142119.1 and negative control were synthesized by QIAGEN (Hilden, Germany). Full-length cDNA of human WDR5 and its truncations were amplified and subcloned into pcDNA3.1 vector. The full-length cDNA of human MYCN was inserted into pcDNA3.1 vector to establish MYCN overexpression vector. Specific siRNAs targeting MYCN or WDR5 and negative control were purchased from Sangon Biotech. Cell transfection was performed with Lipofectamine 2000 (Invitrogen) in line with the manufacturer’s instructions. The sequences of the GapmeRs and siRNAs were presented in Additional file 1: Table S2.
Lentivirus packaging and infection
To generate lentivirus, the lentiviral vectors were co-transfected with packaging plasmids (pMD2G and psPAX2) into HEK293T cells using Lipofectamine 2000 (Invitrogen). The supernatants containing lentiviral particles were harvested at 48 h and 72 h after transfection, which were then concentrated by ultracentrifugation at 120,000 g for 2 h. The NB cells were directly infected with the obtained lentivirus, followed by selecting with puromycin 48 h after infection to produce stable cells.
Protein extraction and western blot assay
Total proteins of NB cells were extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with 1% PMSF. The proteins were quantified by Enhanced BCA Protein Assay Kit (Beyotime), separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% skimmed milk, the bands were incubated with primary antibodies against MYCN (1:1000, CST, Beverly, MA, USA) and GAPDH (1:3000, CST) at 4 ℃ overnight, and then incubated with HRP-conjugated secondary antibodies (1:3000, CST) at room temperature for 2 h. Finally, the bands were visualized with enhanced chemiluminescence system.
Cytoplasmic and nuclear fractionation
The cytoplasmic and nuclear RNA of NB cells were isolated according to the recommended protocols of PARIS™ Kit (Thermo Fisher Scientific). Briefly, the cells were lysed with cell fractionation buffer, followed by low-speed centrifugation to separate the nuclear fraction from the cytoplasmic fraction. The supernatant containing the cytoplasmic fraction was carefully collected and the nuclear pellet was added with cell disruption buffer. Then, the split fractions were mixed with 2 × lysis/binding solution and 100% ethanol and treated with filtration columns. After that, the samples were washed with wash solution and eluted with elution solution.
Fluorescence in situ hybridization (FISH)
FISH assay was performed with Cy3-labeled probes targeting AC142119.1 and Fluorescent In Situ Hybridization kit (RiboBio) following the manufacturer’s instructions. Briefly, the NB cells seeded on the coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After incubating with prehybridization buffer at 37 ℃ for 30 min, the cells were hybridized with hybridization buffer containing specific probes at 37 ℃ overnight. Then, cover slips were washed with 2 × SSC for 6 times and followed by counterstaining with DAPI.
Cell proliferation and cell cycle assays
The proliferation of NB cells was determined by the growth curves generated from Cell Counting Kit-8 (Beyotime) in accordance with the manufacturer’s protocols. For cell cycle analysis, NB cells were collected and treated with 75% ice-cold ethanol. Then, the cells were stained with PI at room temperature for 30 min and analyzed by a flow cytometer.
The four-week-old BALB/c nude mice were purchased from the Center of Experimental Animals of Chongqing Medical University (Chongqing, China) and fed under the standard conditions. Stable SK-N-AS cells (5 × 106) were subcutaneously injected into the dorsal flanks of the randomly grouped nude mice to construct AC142119.1 overexpression xenograft models. As to AC142119.1 knockdown models, SK-N-DZ cells (5 × 106) were inoculated into the mice. When the xenograft tumor grew to 1 mm3, LNA-modified GapmeR targeting AC142119.1 transcript was injected into the tumor of experiment group once every 3 days. The volumes of the tumors were monitored once a week and calculated with the formula, 0.5 × width2 × length. Four weeks later, the tumor-bearing mice were sacrificed and followed by tumor resection and weighing. The animal studies were approved by Chongqing Medical University Animal Care and Use Committee.
For IHC staining, after dewaxed and rehydrated, the paraffin-embedded tissue sections were incubated with primary antibodies against MYCN (1:200, CST) or Ki67 (1:500, CST) at 4 ℃ overnight and biotin-labeled secondary antibodies at 37 ℃ for 1 h. The sections were then stained with DAB and hematoxylin.
RNA immunoprecipitation (RIP)
RIP assay was executed with antibody against WDR5 (CST), IgG control and a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s instruction.
RNA pull-down assay and mass spectrometry
The biotin-labeled full-length and truncated AC142119.1 RNA were transcribed in vitro using T7 RNA polymerase with PCR products used as templates and labeled with RNA 3′ End Desthiobiotinylation Kit (Thermo Fisher Scientific) in accordance with the recommended conditions. RNA pull-down assays were conducted using Pierce™ Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific) according to the standard protocols. In brief, the NB cells were lysed with RIP lysis buffer supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific) and RNase inhibitors, and then incubated with biotin-labeled RNAs combined with streptavidin magnetic beads at 4 ℃ for 1 h. The RNA-binding protein complexes were collected with a magnetic stand and eluted in elution buffer after washing 3 times with wash buffer. The pulled down proteins were analyzed by mass spectrometry at Sangon Biotech or western blot.
RNA electrophoretic mobility shift assay (EMSA)
The biotin-labeled probe specific for AC142119.1 transcript was synthesized by Sangon Biotech. RNA EMSAs were carried out using LightShift® Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, the nuclear extracts of NB cells transfected with WDR5 expression vector or its deletion mutations together with biotin-tagged AC142119.1 probe were incubated in the binding reactions at room temperature for 30 min. Then, the reactions were added with loading buffer and subjected to PAGE using native gels, followed by transferring onto nylon membranes. After crosslinking to the membranes by exposing to UV irradiation, the biotin-labeled probe was detected by streptavidin-HRP conjugate and enhanced chemiluminescence.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were conducted using SimpleChIP® Enzymatic Chromatin IP Kit (CST) with antibodies specific for H3K4me3 (CST) or IgG control. The precipitated DNA fragments were subjected to qRT-PCR analysis.
Chromatin isolation by RNA purification (ChIRP) assay
The biotin-labeled probes targeting AC142119.1 transcript and negative control probes were synthesized by Sangon Biotech. The NB cells were fixed with 1% formaldehyde at room temperature for 10 min and terminated with 2.5 M glycine for 5 min. Then, the cells were lysed and sonicated to shear the chromatin into 100–200 bp fragments. After centrifugation at 12,000 g for 10 min, the supernatant of the sonicated samples was collected and added with 2 × lysate volume of DNA-RNA hybridization buffer, followed by incubating with biotin-labeled probes at room temperature with gentle shaking overnight. Next, the pre-washed streptavidin beads were added into each of the hybridization reaction and incubated at room temperature for 4 h. After the step of 5 times washing, the beads were collected with a magnetic stand and eluted in elution buffer. Finally, the bound RNA and DNA fragments were purified from the beads.
All the statistical analyses were conducted with SPSS 21.0. Differences between two or more groups were assessed by student’s t-test or one-way ANOVA, respectively. The relevance between two groups were determined by Pearson correlation. P < 0.05 was considered as statistically significant.
Identification of the MYCN-related lncRNA AC142119.1
In order to identify the lncRNAs that are important for MYCN amplification-induced tumorigenesis of NB, we analyzed the lncRNA expression profile using microarray in MYCN-amplified cell line SK-N-DZ and MYCN-non-amplified cell line SH-SY5Y. Differential expression analysis indicated that 1608 lncRNAs were upregulated and 1647 lncRNAs were downregulated in MYCN-amplified SK-N-DZ cells with the cut-off criteria of fold change > 2 and P < 0.05 (Fig. 1A), including lncNB1 (also known as RP1-40E16.9), AF127936.7 and RP11-102F4.3 that have been previously reported to be significantly increased in MYCN-amplified NB cell lines, which proved the robustness of our data to a certain extent . Considering that lncRNAs are known to modulate their neighboring protein-coding genes in cis. We identified three lncRNA candidates (AC142119.1, AC010145.3 and AC130710.1) of these differentially expressed lncRNAs that were located nearby the MYCN locus. To prioritize the most MYCN-relevant and biologically significant lncRNA, we concentrated on AC142119.1, as it was the most upregulated one (16.52 folds) in MYCN-amplified SK-N-DZ cell line and resided upstream of MYCN locus (Fig. 1B, C). According to the annotation of UCSC database (http://genome.ucsc.edu/), AC142119.1 was a lncRNA containing 3 exons located at chr2: 16096426-16105662 and predicted to produce two transcripts with length of 853nt and 642nt, respectively. To confirm the full-length transcripts of AC142119.1 in NB cells, both 5′ RACE and 3′ RACE assays were executed and determined a novel isoform of AC142119.1 with a length of 924nt in SK-N-DZ cells (Fig. 1D and Additional file 2: Fig. S1A), which was discordant with the prediction of UCSC database. Subsequent analyses of the novel AC142119.1 transcript using PRIDE, PhyloCSF, CPAT, Bazzini small ORFs and Lee translation initiation sites revealed the low coding potential of AC142119.1, corroborating the identity of AC142119.1 as a non-coding gene (Additional file 2: Fig. S1B). Cellular fractionation assays followed by qRT-PCR analysis disclosed that AC142119.1 was predominately localized in the nucleus of SK-N-DZ cells with a small proportion distributing in the cytoplasm (Fig. 1E), which was further substantiated by FISH assay with specific probes (Fig. 1F). All the above data suggested that lncRNA AC142119.1 was a novel non-coding gene and highly expressed in MYCN-amplified NB cells.
AC142119.1 is elevated in MYCN-amplified NB tissues and positively correlated with the poor outcome in NB patients
In order to determine the expression pattern of AC142119.1 in NB, qRT-PCR was performed in two MYCN-amplified (SK-N-DZ and IMR-32 cells) and non-amplified (SH-SY5Y and SK-N-AS cells) NB cell lines. As a result, cells with MYCN amplification showed considerably higher expression levels of AC142119.1 than those without MYCN amplification (Fig. 2A). Meanwhile, MYCN-amplified NB cell lines also expressed high levels of MYCN mRNA and protein compared with MYCN-non-amplified cell lines (Fig. 2B, C). Next, we examined the expression levels of AC142119.1 in randomly collected 55 NB tissues, finding that NB patients with advanced INSS stage and high risk showed much higher expression levels of AC142119.1 than those in control groups (Fig. 2D, E). Consistently, MYCN exhibited analogous expression patterns with AC142119.1 in NB tissues (Fig. 2F, G). Moreover, Pearson correlation analysis revealed a strong positive correlation between AC142119.1 and MYCN mRNA levels in these NB tissues (Fig. 2H). Importantly, Kaplan–Meier survival curve showed that the expression level of AC142119.1 was negatively correlated with the overall survival of NB patients (Fig. 2I). These findings established that AC142119.1 was positively correlated with MYCN and may contribute to the progression of NB.
AC142119.1 facilitates the proliferation of NB cells in vitro
To investigate the biological role of AC142119.1 in NB, we stably overexpressed AC142119.1 in SK-N-AS and SH-SY5Y, two cell lines with low level of AC142119.1, by infecting with lentivirus carrying AC142119.1, which was confirmed by qRT-PCR (Fig. 3A). Given that AC142119.1 was localized in the nuclear of NB cells, LNA-modified antisense oligonucleotides (GapmeRs) were applied in SK-N-DZ and IMR-32 cells with high level of AC142119.1 to knock down AC142119.1 (Fig. 3B). Then, we detected the viability of the NB cells using CCK-8 assays, finding that enforced expression of AC142119.1 enhanced the proliferation of SK-N-AS and SH-SY5Y cells (Fig. 3C), whereas silencing of AC142119.1 inhibited the growth of SK-N-DZ and IMR-32 cells (Fig. 3D). In addition, we also assessed whether AC142119.1 affects the cell cycle of NB cells. As anticipated, SK-N-AS and SH-SY5Y cells with overexpressed AC142119.1 had larger proportion of S phase and smaller proportion of G1 phase than those in control group by flow cytometry analyses (Fig. 3E), while deficiency of AC142119.1 produced an entirely opposite effect in SK-N-DZ and IMR-32 cells, implying the proliferation-promoting effect of AC142119.1 on NB cells (Fig. 3F). Collectively, these results substantiated that AC142119.1 acted as an oncogenic driver in NB.
AC142119.1 contributes to the tumorigenesis of NB in vivo
To further probe the effect of AC142119.1 on tumorigenesis of NB in vivo, SK-N-AS cells with stable overexpression of AC142119.1 and control cells were subcutaneously inoculated into the dorsal flank of nude mice to construct xenograft models. We observed that enforced expression of AC142119.1 significantly accelerated the formation and growth of xenograft tumors (Fig. 4A–C). Furthermore, we also established AC142119.1 knockdown xenograft models by inoculating the mice with SK-N-DZ cells and treating the tumors with LNA-modified GapmeR. Four weeks later, tumors treated with LNA-modified GapmeR specific for AC142119.1 had much smaller volumes and lower weights than those in control group, pinpointing that depletion of AC142119.1 was unfavorable for the growth of xenograft tumors (Fig. 4D–F). To validate the regulatory role of AC142119.1 on MYCN in vivo, we analyzed the expression of MYCN in these xenograft tumors. qRT-PCR analyses uncovered that the mRNA level of MYCN were significantly increased in AC142119.1 overexpression group while decreased in the AC142119.1 knockdown group (Fig. 4G, H). Similarly, IHC staining indicated that tumors with overexpressed AC142119.1 had higher MYCN and Ki67 protein levels than control group, whereas downregulation of AC142119.1 generated a reverse effect (Fig. 4I and Additional file 3: Fig. S2). These data demonstrated that AC142119.1 facilitates the tumorigenesis of NB in vivo.
AC142119.1 directly binds with WDR5 in NB cells
The above findings impelled us to explore the underlying mechanisms by which AC142119.1 regulates the expression of MYCN in NB. Given that lncRNAs usually cooperated with specific proteins, such as chromatin-modifying complexes, transcriptional factors and RNP complexes, to conduct their regulatory roles . Therefore, we performed RNA pull-down assay with in-vitro-transcribed biotinylated AC142119.1 RNA to identify the protein partners of AC142119.1 transcript in NB cells. The captured proteins were separated by SDS-PAGE and subjected to silver staining (Fig. 5A). Following mass spectrometry analysis identified 594 unique proteins were specifically enriched by AC142119.1 RNA, rather than the antisense AC142119.1 transcript. Among these proteins, WD repeat-containing protein 5 (WDR5) attracted our attention, which was a key component of histone H3K4 methyltransferase complex, and could catalyze H3K4me3 . It is well-known that H3K4me3 is associated with the transcriptional activation of target genes . Moreover, previous study demonstrated that WDR5 served as a tumor-promoter in NB and high level of WDR5 was positively correlated with poor prognosis of NB patients . Therefore, we hypothesized that AC142119.1 might cooperate with WDR5 to increase the H3K4me3 level and activate the transcription of MYCN gene in NB. We then validated the ability of WDR5 to bind with AC142119.1 by western blot in the retrieved proteins from the RNA pull-down assay (Fig. 5B). Subsequent RIP assays performed with antibodies specific for WDR5 further consolidate the interaction between the endogenous AC142119.1 transcript and WDR5 protein in SK-N-DZ and IMR-32 cells (Fig. 5C). We then explored the detailed interaction region between AC142119.1 transcript and WDR5 protein. By using catRAPID (http://s.tartaglialab.com/page/catrapid_group), an algorithm to evaluate the binding propensity of protein-RNA pairs, we found that two fragments of AC142119.1 RNA (1-400nt and 599-850nt) showed high affinity with WDR5 protein (Fig. 5D, E). Based on this prediction, biotin-tagged AC142119.1 RNA truncations were constructed and used as probes for RNA pull-down assays (Fig. 5F). The results showed that both AC142119.1-Δ1 and AC142119.1-Δ2 could bind with WDR5 protein, whereas AC142119.1-Δ3 was unable to interact with WDR5, implying the 1-400 bp of AC142119.1 transcript was critical for the interaction with WDR5 in NB cells (Fig. 5G). To further determine the region of WDR5 bound with AC142119.1 RNA, the truncated forms of WDR5 (WDR5-D1, WDR5-D3, WDR5-D6 and WDR5-7) were also established and applied to RNA EMSA along with biotin-labeled AC142119.1 probes in NB cells. We found that the domains of WDR5-D1 and WDR5-D7, rather than other domains, were indispensable for the interaction between WDR5 and AC142119.1 RNA (Fig. 5H). Collectively, these findings proposed that AC142119.1 could directly bind with WDR5 in NB cells.
AC142119.1 recruits WDR5 protein to activate the transcription of MYCN
To further validate our hypothesis, we performed ChIRP assay with biotin-labeled probes specific for AC142119.1 in SK-N-DZ cells to observe the association between the AC142119.1 transcript and MYCN promoter. As a result, we found that AC142119.1 was significantly enriched at the regions of − 2000 – − 1800 bp and − 600 – − 400 bp from the transcription start site of MYCN promoter (Fig. 6A). In addition, the results of ChIP assays revealed that enforced expression of AC142119.1 could markedly increase the histone active modification H3K4me3 of MYCN promoter while knockdown of AC142119.1 generated an opposite effect (Fig. 6B). These data suggested that AC142119.1 could recruit WDR5 to the promoter region of MYCN and enhance the H3K4me3 level of MYCN promoter. Next, we analyzed the effect of AC142119.1 on the expression of MYCN in NB cells. Notably, both of the results of qRT-PCR and western blot assays showed that ectopic expression or depletion of AC142119.1 resulted in significant increase or decrease of MYCN at both mRNA and protein levels, respectively (Fig. 6C, D). However, overexpression or knockdown of MYCN had no effect on AC142119.1 expression levels in NB cells (Additional file 4: Fig. S3).
AC142119.1 promotes the progression of NB through interacting with WDR5
To understand whether AC142119.1 exerts its oncogenic role via WDR5 in NB, we conducted rescue experiments in NB cell lines. CCK-8 assays indicated that knockdown of WDR5 significantly abolished the increase of cell growth induced by overexpression of AC142119.1 in SH-SY5Y and SK-N-AS cells (Fig. 7A), whereas ectopic expression of WDR5 could reverse the proliferation-suppressing effect produced by downregulation of AC142119.1 in SK-N-DZ and IMR-32 cells (Fig. 7B). Consistently, cell cycle analyses showed that knockdown or overexpression of WDR5 abolished the promotive or inhibitory effects on cell cycle progression induced by upregulation or downregulation of AC142119.1 in NB cells (Fig. 7C, D). Moreover, we also performed western blot analyses and found that the expression level of MYCN was upregulated or downregulated after ectopic expression or knockdown of AC142119.1 in NB cells, which was rescued by silencing or overexpression of WDR5 (Fig. 7E, F). Altogether, the above findings suggested that AC142119.1 recruited WDR5 protein to activate the transcription of MYCN and further promoted the progression of NB (Fig. 8).
Amplification of oncogene MYCN is considered as a major driver of NB and one of the strongest predictors of poor clinical outcomes of NB patients . Although MYCN amplification or overexpression could be detected in approximately half of all high-risk NB cases, the molecular mechanism underlying the ectopic expression of MYCN has not been fully elucidated . Recent years, lncRNAs have emerged as oncogenes or tumor suppressors in the initiation and progression of various human cancers, including NB [28,29,30]. However, whether individual lncRNAs contribute to the ectopic expression of MYCN in NB is largely unknown.
In the current study, by using microarray analysis, we firstly identified a novel MYCN-related lncRNA termed as AC142119.1, which was significantly elevated in MYCN-amplified NB cell lines and tissues, as well as NB tissues with advanced INSS stage and high risk. In addition, Pearson correlation analysis revealed a strong positive correlation between the expression of AC142119.1 and MYCN mRNA in NB tissues. Importantly, NB patients with higher expression of AC142119.1 had shorter overall survival time than those with low level of AC142119.1. By utilizing both loss- and gain-of-function approaches, we demonstrated that AC142119.1 was an onco-lncRNA that could promote the proliferation and cell cycle progression of NB cell lines in vitro and accelerate tumor growth in vivo. Our findings uncovered the oncogenic role of AC142119.1 in the progression of NB, supporting the possibility of AC142119.1 as a promising predictor of malignant progression and potential therapeutic target for NB.
Amounting evidence demonstrated that lncRNAs could exert their regulatory role on neighboring protein-coding genes in cis by diverse mechanisms. For example, lncRNA transcript RBAT1 recruits hnRNPL to the promoter of oncogene E2F3 to ci-activate its transcription, resulting in the tumorigenesis of retinoblastoma . Moreover, lncRNA TMPO-AS1 recruited FUS and p300 to the promoter of TMPO and formed a complex in situ, which activated the transcription of TMPO gene through increasing the H3K27ac level in its promoter . In addition, lncRNA RUNXOR regulates the expression of RUNX1 by altering the spatial structure of chromatin and enhancing histone H3K4me3 levels of RUNX1 promoter . In our study, to ascertain whether lncRNA could modulate the expression of MYCN in NB in cis, we sought out three differentially expressed lncRNA candidates (AC142119.1, AC010145.3 and AC130710.1) located in the upstream or downstream of MYCN gene locus by bioinformatics analysis. Moreover, we found that none of them has been reported in cancers or other human diseases. AC142119.1, as the most upregulated one in MYCN-amplified NB cells, was then chosen for the further investigation. According to UCSC database, AC142119.1 was predicted to produce two transcripts with length of 853nt and 642nt, respectively. Here, we presented a novel isoform of AC142119.1 with a length of 924nt in SK-N-DZ cells. Moreover, we observed that AC142119.1 transcript predominantly localized in the nucleus of NB cells and significantly correlated with the expression of MYCN mRNA in NB tissues. Our findings preliminarily testified that AC142119.1 might work in cis to regulate the expression of MYCN in NB.
Our subsequent data of biotin-labeled RNA pull-down, RIP and RNA EMSA assays demonstrated that AC142119.1 transcript could directly interact with WDR5. It has been well established that the highly-conserved nuclear protein WDR5 participates in the formation of multiple chromatin-modifying complexes, including SET1/MLL histone methyltransferase which catalyzes the trimethylation of histone H3K4 . As a critical subunit of SET1/MLL complex, WDR5 promotes the assembly and activity of the complex by binding to the arginine-bearing motif of MLL . SET1/MLL complexes mediated H3K4me3 is one of the most important histone modifications and usually selectively localizes to the promoter region and downstream transcription start sites to activate the transcription of target genes . Previous study revealed that WDR5-H3K4me3 epigenetic axis could modulate the expression of OPN to enhance the immune escape of pancreatic cancer . Additionally, WDR5 promotes the cell proliferation, self-renewal and chemoresistance of bladder cancer by activating multiple oncogenes via increasing H3K4me3 levels . Under hypoxic conditions, WDR5 is upregulated and binds with histone deacetylase HDAC3, leading to the increased levels of histone H3K4me3 and overexpression of mesenchymal genes . More importantly, in NB, WDR5 was positively associated with the expression of MYCN and formed a protein complex with MYCN to induce H3K4me3 and transcriptional activation of MDM2 gene, thus resulting in the initiation and progression of NB . However, WDR5 protein itself has no putative DNA-binding domains, and the mechanism of how WDR5 locates to target sites in chromatin is not certain.
Recent studies indicated that several lncRNAs, such as TM4SF19-AS1, BLACAT2 and SATB2-AS1 and PTPRE-AS1, could directly interact with WDR5 protein and serve as a guide to recruit chromatin regulatory complexes to target gene locus [40,41,42,43]. Consistent with the former studies, we proved that AC142119.1 transcript could not only bind with WDR5 protein, but also directly interact with the promoter region of MYCN. More importantly, ChIP assays revealed that the H3K4me3 levels of MYCN promoter was significantly increased or decreased after overexpression or knockdown of AC142119.1 in NB cells, meanwhile, the mRNA and protein levels of MYCN are correspondingly enhanced or inhibited. Importantly, manipulation of the expression of MYCN did not affect the expression level of AC142119.1, implying that AC142119.1 is indeed an upstream regulator of MYCN gene. Our data suggests that AC142119.1 activates the transcription of MYCN by recruiting WDR5 protein to the promoter of MYCN through the formation of a ternary complex with target DNA sequence, and increasing the H3K4me3 level of MYCN promoter in NB cells. However, further investigation was needed to fully understand the biological function and detailed mechanisms of AC142119.1 in NB.
In summary, our findings revealed that AC142119.1 is an oncogenic lncRNA that is significantly elevated in NB tissues with MYCN amplification, advanced INSS stage or high risk. Moreover, AC142119.1 serves as an epigenetic modifier to activate the transcription of MYCN gene by recruiting WDR5 protein to the promoter of MYCN and increasing its H3K4me3 levels, eventually leading to the malignant progression of NB. Therefore, our study provides new insights into the underlying mechanism of the ectopic expression of MYCN in NB and raises the possibility of AC142119.1 to serve as a biomarker for the progression assessment and a therapeutic target for NB.
Availability of data and materials
The data supporting the results of the study were available from the corresponding author upon reasonable request.
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This study was partially supported by Natural Science Foundation of China (82070167, 82270160), the Chongqing Bureau of Science and Technology and Chongqing Municipal Health Commission joint research project (2021MSXM167), the General Basic Research Project from the Ministry of Education Key Laboratory of Child Development and Disorders (GBRP-202103) and Chongqing Graduate Scientific Research Innovation Project (CYB19154).
Ethics approval and consent to participate
The present study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Chongqing Medical University (No.2021-191). Animal studies were approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University (No.2022-224).
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The sequences of primers used in the current study. Table S2. The sequences of GapmeRs and siRNAs used in the current study.
The identification of AC142119.1 transcript. A AC142119.1 transcript validated by RACE assays in SK-N-DZ cells. B The coding potential of AC142119.1 was assessed by PRIDE, PhyloCSF, CPAT, Bazzini small ORFs and Lee translation initiation sites, respectively.
The expression level of Ki67 in xenograft tumors was determined by IHC staining. Scale bar, 100 μm.
The expression of AC142119.1 was detected by qRT-PCR after overexpression or knockdown of MYCN in NB cells. ns, no significance.
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Yang, R., Liu, N., Li, T. et al. LncRNA AC142119.1 facilitates the progression of neuroblastoma by epigenetically initiating the transcription of MYCN. J Transl Med 21, 659 (2023). https://doi.org/10.1186/s12967-023-04535-3