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Song et al. Journal of Hematology & Oncology(2020) 13:24Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assay was performed according to instructions of EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [9, 11, 14], usingantibodies specific for HNF4A (ab181604), MYCN(ab16898), CTCF (ab188408), RNA polymerase II (RNAPol II, ab5131), histone H3 lysine 4 trimethylation(H3K4me3, ab8580), and histone H3 lysine 27 trimethylation (H3K27me3, ab6002). Real-time quantitative PCR(qPCR) was performed with SYBR Green PCR MasterMix (Applied Biosystems) and primers (Additional file 1:Table S1). Immunoprecipitated DNA was normalized toinput DNA, using isotype IgG as a negative control.Gene over-expression and knockdownHuman HNF4A and MYCN expression vectors wereprovided by Dr. David Martinez Selva [16] and Dr.Arturo Sala [17], respectively. Human HNF4A-AS1cDNA (648 bp), hnRNPU cDNA (2478 bp), CTCF cDNA(2184 bp), and their truncations were amplified from NBtissues (Additional file 1: Table S2) and subcloned intopcDNA3.1 (Invitrogen), pCMV-3Tag-1C, pCMV-NMyc, and pGEX-6P-1 (Addgene, Cambridge, MA), respectively. Mutation of short hairpin RNA (shRNA)-targeting site of HNF4A-AS1 or hnRNPU RGG residueswas performed with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) and primers (Additional file1: Table S2). Oligonucleotides encoding shRNAs specificfor HNF4A, HK2, SLC2A1, MYCN, HNF4A-AS1,hnRNPU, or CTCF (Additional file 1: Table S3) weresubcloned into GV298 (Genechem Co., Ltd, Shanghai,China). Single guide RNAs (sgRNAs) targeting downstream region of HNF4A-AS1 transcription start site(Additional file 1: Table S3) were inserted into dCas9BFP-KRAB (Addgene). Stable cell lines were screened byadministration of neomycin or puromycin (Invitrogen).Rescue of target gene expressionTo rescue gene expression altered by hnRNPU, CTCF,or HNF4A knockdown, tumor cells were transfectedwith HNF4A-AS1 vector. To restore target gene expression induced by over-expression of hnRNPU, CTCF, orHNF4A, shRNA specific for HNF4A-AS1 (Additional file1: Table S3) was transfected into tumor cells with Genesilencer Transfection Reagent (Genlantis, San Diego,CA). Empty vector and sh-Scb were applied as controls(Additional file 1: Table S3).Lentiviral packagingLentiviral vectors were co-transfected with packagingplasmids psPAX2 and pMD2G (Addgene) intoHEK293T cells. Infectious lentivirus was filtered through0.45 μm PVDF filters, and concentrated 100-fold byultracentrifugation (2 h at 120,000g). Infectious lentivirusPage 3 of 19was harvested at 36 and 60 h after transfection and filtered through 0.45 μm PVDF filters. Recombinant lentivirus was concentrated 100-fold by ultracentrifugation(2 h at 120,000g). Lentivirus-containing pellet was dissolved in phosphate buffer saline (PBS) and injected inmice within 48 h.Rapid amplification of cDNA ends assayTotal RNA was isolated from BE(2)-C cells to prepare rapidamplification of cDNA ends (RACE)-ready cDNA usingSMARTer RACE cDNA Amplification Kit (Clontech, PaloAlto, CA), which was further amplified by PCR primers andnested PCR primers (Additional file 1: Table S1).Northern blotThe 254-bp probe was in vitro transcribed using DIGLabeling Kit (MyLab Corporation, Beijing, China) andT7 RNA polymerase, and treated with RNase-free DNaseI. For Northern blot, 20 μg of total RNA was separatedon 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered 2% (w/v) agarose gel containing 1.2% (v/v) formaldehyde under denaturing conditions for 4 h at 80 V, andtransferred to Hybond-N membrane (Pall Corp., PortWashington, NY). Prehybridization was carried out at65 C for 30 min in DIG Easy Hyb solution (Roche, Indianapolis, IN). Hybridization was performed at 65 Cfor 16–18 h. Blots were washed stringently, detected byanti-digoxigenin (DIG) antibody, and recorded on X-rayfilms with chemiluminescence substrate CSPD (Roche).RNA fluorescence in situ hybridizationAntisense or sense RNA probe for HNF4A-AS1 wasin vitro transcribed with Biotin RNA Labeling Mix(Roche) and T7 RNA polymerase. Cells were seeded oncoverslips, fixed in 4% paraformaldehyde for 15 min, andincubated with 40 nmol·L 1 fluorescence in situhybridization (FISH) probe in hybridization buffer (100mg/ml dextran sulfate, 10% formamide in 2 SSC) at80 C for 2 min. Hybridization was performed at 55 Cfor 2 h, with or without RNase A (20 μg) treatment. Cellswere incubated with streptavidin-conjugated fluoresceinisothiocyanate (FITC), with nuclei counterstained with4′,6-diamidino-2-phenylindole (DAPI).Fluorescence immunocytochemical stainingCells were plated on coverslip, incubated with 5% milkfor 1 h, and treated with antibody specific for hnRNPU(ab10297, Abcam Inc., 1:300 dilution) at 4 C overnight.Then, coverslips were treated with Alexa Fluor 594 goatanti-rabbit IgG (1:1000 dilution) and stained with DAPI(300 nmol·L 1). The images were photographed under aNikon A1Si Laser Scanning Confocal Microscope(Nikon Instruments Inc, Japan).

Song et al. Journal of Hematology & Oncology(2020) 13:24RNA sequencingTotal RNA of tumor cells (1 106) was isolated usingTRIzolTM reagent (Life Technologies, Inc., Gaithersburg,MD). Library preparation and transcriptome sequencingon an Illumina HiSeq X Ten platform were carried outat Novogene Bioinformatics Technology Co., Ltd.(Beijing, China) to generate 100-bp paired-end reads.HTSeq v0.6.0 was applied in counting the numbers ofread mapping to each gene, and fragments per kilobaseof transcript per million fragments mapped (FPKM) ofeach gene were calculated. Sequencing results have beendeposited in GEO database (accession code GSE143896).Page 4 of 19mobility shift assay (EMSA) using nuclear extracts or recombinant hnRNPU protein was performed using LightShift Chemiluminescent RNA EMSA Kit (Thermo FisherScientific, Inc.).Co-immunoprecipitation assayCo-immunoprecipitation (Co-IP) was performed as previously described [9, 12, 14], with antibodies specific forhnRNPU (ab10297), CTCF (ab188408), FLAG(ab125243), or Myc (ab9106, Abcam Inc.). Bead-boundproteins were released and analyzed by Western blot.Bimolecular fluorescence complementation assayBiotin-labeled RNA pull-down and mass spectrometryanalysisBiotin-labeled RNA probes for HNF4A-AS1 truncationswere in vitro transcribed as described above. Nuclear extracts were harvested, resuspended in freshly preparedproteolysis buffer, and incubated with biotin-labeledRNA (10 pmol) and streptavidin-agarose beads (Invitrogen) for 1 h. Precipitated components were separatedusing SDS-PAGE, followed by Coomassie blue stainingor Western blot. Differential bands were harvested formass spectrometry analysis (Wuhan Institute of Biotechnology, Wuhan, China) [9].Cross-linking RNA immunoprecipitationTumor cells (1 108) were ultraviolet light cross-linkedat 254 nm (200 J/cm2) in PBS and collected by scraping[9, 13]. RNA immunoprecipitation (RIP) assay was performed with Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA), usingantibodies for hnRNPU (ab10297) or hnRNPK (ab39975,Abcam Inc.). Co-precipitated RNAs were detected byRT-PCR or real-time quantitative qRT-PCR withprimers (Additional file 1: Table S1). Total RNAs (input)and isotype antibody (IgG) were applied as controls.In vitro binding assayHuman hnRNPU cDNA (2478 bp) and CTCF cDNA(2184 bp) were subcloned into bimolecular fluorescencecomplementation (BiFC) vectors pBiFC-VN173 andpBiFC-VC155 (Addgene) and co-transfected into tumorcells with Lipofectamine 2000 (Invitrogen) for 24 h.Fluorescence emission was observed under a confocalmicroscope, using excitation and emission wavelengthsof 488 and 500 nm, respectively [14].Design and synthesis of inhibitory peptidesInhibitory peptides for blocking interaction betweenHNF4A-AS1 and hnRNPU were designed. The 11 aminoacid long peptide (YGRKKRRQRRR) from Tat proteintransduction domain served as a cell-penetrating peptide. Thus, inhibitory peptides were chemically synthesized by linking with biotin-labeled cell-penetratingpeptide at N-terminus and conjugating with FITC at Cterminus (ChinaPeptides Co. Ltd, Shanghai, China), withpurity larger than 95%.Biotin-labeled peptide pull-down assayCellular proteins were isolated using 1 cell lysis buffer(Promega), and incubated with biotin-labeled peptideand streptavidin-agarose at 4 C. Then, incubation of celllysates with streptavidin-agarose was undertaken at 4 Cfor 2 h. Beads were extensively washed, and RNA pulleddown was measured by RT-PCR or real-time qRT-PCR.A series of hnRNPU truncations were amplified fromNB tissues (Additional file 1: Table S2), subcloned intopGEX-6P-1 (Addgene), and transformed into E. coli toproduce GST-tagged hnRNPU [9, 11]. HNF4A-AS1cRNA was in vitro transcribed with TranscriptAid T7High Yield Transcription Kit (Thermo Fisher Scientific,Inc., Waltham, MA), and incubated with GST-taggedhnRNPU. HnRNPU–RNA complexes were pulled downusing GST beads (Sigma). Protein was detected by SDSPAGE and western blot, while RNA was measured byRT-PCR with primers (Additional file 1: Table S1).Cellular glucose uptake, lactate production, and ATPlevels were detected as previously described [18]. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured in XF media under basalconditions and in response to glucose (10 mmol·L 1), oligomycin (2 μmol·L 1), and 2-deoxyglucose (100mmol·L 1), using a Seahorse Biosciences XFe24 FluxAnalyzer (North Billerica, MA).RNA electrophoretic mobility shift assayCellular viability, growth, and invasion assaysBiotin-labeled RNA probes for HNF4A-AS1 truncationswere prepared as described above. RNA electrophoreticThe 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) colorimetric [9, 19], softAerobic glycolysis and seahorse extracellular flux assays

Song et al. Journal of Hematology & Oncology(2020) 13:24Page 5 of 19agar [9, 11, 12, 14, 20], and matrigel invasion [9, 11,12, 14, 20, 21] assays for measuring the viability,growth, and invasion capability of tumor cells wereconducted as previously described.between two gene lists. Log-rank test was used to assesssurvival difference. All statistical tests were two-sidedand considered statistically significant when false discovery rate (FDR)-corrected P values were less than 0.05.In vivo tumorigenesis and aggressiveness assaysResultsAll animal experiments were carried out in accordancewith NIH Guidelines for the Care and Use of LaboratoryAnimals, and approved by the Animal Care Committeeof Tongji Medical College (approval number,Y20080290). In vivo tumor growth and experimentalmetastasis studies were performed with blindly randomized 4-week-old female BALB/c nude mice (n 5 pergroup) as previously described [9, 11, 12, 14, 20]. Forin vivo therapeutic studies, tumor cells (1 106 or 0.4 106) stably expressing red fluorescent protein wereinjected into dorsal flanks or tail vein of nude mice, respectively. One week later, mice were blindly randomized and treated by tail vein injection of synthesized cellpenetrating peptide [22] or lentivirus-mediated shRNA(1 107 plaque-forming units in 100 μl PBS), and imaged using In-Vivo Xtreme II small animal imaging system (Bruker Corporation, Billerica, MA).Transcription factor HNF4A facilitates glycolytic geneexpression and glycolysis of NB cellsPatient tissue samplesThe Institutional Review Board of Tongji Medical Collegeapproved human tissue study (approval number, 2011S085). All procedures were carried out in accordance withguidelines set forth by Declaration of Helsinki. Written informed consent was obtained from all legal guardians ofpatients. Patients with a history of preoperative chemotherapy or radiotherapy were excluded. Human normaldorsal root ganglia tissues were collected from therapeuticabortion. All fresh specimens were frozen in liquid nitrogen, validated by pathological diagnosis, and stored at 80 C until use. Blood samples were centrifuged at 2000 r/min for 10 min at 4 C, and serum was collected andstored at 80 C until further processing.ImmunohistochemistryImmunohistochemical staining and quantitative evaluationwere performed as previously described [9, 11, 12, 14, 20],with antibody specific for Ki-67 (ab92742, Abcam Inc.; 1:100dilution) or CD31 (ab28364, Abcam Inc.; 1:100 dilution).The degree of positivity was measured according to percentage of positive tumor cells.Statistical analysisAll data were shown as mean standard error of themean (s.e.m.). Cutoff values were determined by averagegene expression levels. Student’s t test, analysis of variance (ANOVA), and χ2 analysis were applied to comparedifference in tumor cells or tissues. Fisher’s exact testwas applied to analyze statistical significance of overlapComprehensive analysis of a public dataset (GSE45547)[23] of 649 NB cases identified 21 and 18 glycolyticgenes differentially expressed (P 0.05) in NB specimenswith varied international neuroblastoma staging system(INSS) stages or associated with survival of patients, respectively (Fig. 1a). Over-lapping analysis (P 0.001) revealed that 15 glycolytic genes were consistentlyassociated with advanced INSS stages and survival of NB(Fig. 1a). Similarly, we found 330 transcription factorsconsistently associated with these clinical features of NBcases, which were subjective to further over-lapping analysis with 36 transcription factors regulating 15 glycolyticgenes analyzed by ChIP-X program [24]. The results indicated that 5 transcription factors might regulate theexpression of glycolytic genes (Fig. 1a). Among them,the activity of HNF4A was most significantly elevated inSH-SY5Y cells in response to treatment with insulin, aninducer of aerobic glycolysis [25] (Additional file 1: Figure S1a). In NB tissues and cell lines, P1 promoterderived HNF4A transcripts were upregulated, while P2HNF4A was expressed at very low levels (Additional file1: Figure S1b). By using isoform-specific primers, endogenous expression of α1-α3 isoforms (especially highlevels of α1 isoform) of P1-HNF4A (referred as HNF4A)was noted in NB tissues, without detectable levels of α4α6 isoforms (Additional file 1: Figure S1c). In addition,high HNF4A protein levels were noted in NB cell lines(Additional file 1: Figure S1b). Stable over-expression orknockdown of HNF4A increased and decreased thelevels of HK2 or SLC2A1, but not of fructosebisphosphate B (ALDOB), lactate dehydro

(Roche, Indianapolis, IN). PCR and real-time PCR were performed with Taq PCR Master Mix or SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and primers (Additional file 1: Table S1). For real-time qRT-PCR assay, the transcript levels were analyzed by 2 method. Western blot Tissue or cellular protein was extracted with 1 cell