Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 is upregulated and facilitates fatty acid oxidation and mitochondrial activity in NB. a Western blot assay showing the p113 levels in normal dorsal ganglia (DG), tumor (T) tissues of NB, and cultured tumor cell lines. b Western blot assay indicating the expression of p113 and CUX1 isoforms (p200 and p110) in SH-SY5Y, SH-N-SH, BE(2)-C, and IMR32 cells stably transfected with empty vector (mock), ecircCUX1 , ecircCUX1 with ORF mutation ( ecircCUX1 Mut), scramble shRNA (sh-Scb), or sh-ecircCUX1. c Western blot assay showing the cytoplasmic and nuclear accumulation of p113 in SH-SY5Y cells stably transfected with mock or p113 . d Immunofluorescence assay revealing cytoplasmic and nuclear localization of p113 in SH-SY5Y and SK-N-SH cells stably transfected with Flag-tagged p113 (upper panel), and those in BE(2)-C and IMR32 cells (lower panel), with nuclei stained by DAPI (blue). Scale bar: 10 μm. e and f Heatmap (e) and quantification (f) of metabolite profiling assay indicating the fatty acid levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 3). g Seahorse extracellular flux assay showing the oxygen consumption rate (OCR) levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1, and those treated with BSA or oleic acid (OLE, 200 μmol·L − 1 , n = 4). h Relative NAD + /NADH ratio and ATP levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1, and those treated with BSA or OLE (200 μmol·L − 1 , n = 5). ANOVA compared the difference in f - h . * P < 0.05 vs. mock, sh-Scb, or sh-Scb + BSA. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in a - d , g and h

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Activity Assay, Western Blot, Cell Culture, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, shRNA, Immunofluorescence, Staining, XF Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 physically interacts with ZRF1 and BRD4 in NB cells. a Volcano plots showing differentially expressed genes (fold change> 1.5, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 . b Coomassie blue staining (left panel) and Venn diagram (right panel) revealing identification of p113-interacting proteins pulled down by p113 or Flag-tag antibody in SH-SY5Y cells stably transfected with 3Flag-tagged p113 , and those overlapped with transcription factors (TF) or epigenetic factors derived from ChIP-X and EpiFactors databases. c Co-IP and western blot assays indicating the interaction among p113, ZRF1, and BRD4 in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , scramble shRNA (sh-Scb), or sh-ecircCUX1. d Secondary co-IP assays showing protein interaction among p113, ZRF1, and BRD4 in SH-SY5Y cells stably transfected with HA-tagged p113 , Flag-tagged ZRF1 , and His-tagged BRD4 . e BiFC assay revealing the interaction of p113 with ZRF1 or BRD4 (arrowheads) in SH-SY5Y cells stably transfected with indicated constructs, with nuclei stained by DAPI. Scale bars: 10 μm. f and g Western blot assay (g) validating the knockdown of ZRF1 or BRD4 in SH-SY5Y cells stably transfected with scramble (Scb) or specific sgRNA for CRISPR interference (CRISPRi, f). Wild type (WT) cells were taken as negative controls. h Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with CRISPRi sgRNA specific against ZRF1 or BRD4 . i Schematic illustration of protein interaction among p113, ZRF1, and BRD4. j Dual-luciferase assay showing the activity of ZRF1 in NB cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 5). Fisher’s exact test for overlapping analysis in b . ANOVA compared the difference in j . * P < 0.05 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c - e , g , h and j

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, FLAG-tag, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, shRNA, Bimolecular Fluorescence Complementation Assay, Construct, CRISPR, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113/ZRF1/BRD4 complex promotes lipid metabolic reprogramming and mitochondrial complex I activity in NB cells. a Heatmap, distribution, and binding motif of ChIP-Seq (left panel) assay revealing genomic enrichment of ZRF1 in SH-SY5Y cells, while Venn diagram, heatmap, and GO pathway (right panel) showing identification of p113/ZRF1/BRD4 target genes by overlapping analysis of RNA-seq results upon p113 over-expression and ChIP-seq peaks of ZRF1 or BRD4. b ChIP-seq assay showing the binding peak of BRD4 or ZRF1 on promoter regions of ALDH3A1 , NDUFA1 , or NDUFAF5 in SH-SY5Y cells. c Western blot assay indicating the expression of ALDH3A1, NDUFA1, or NDUFAF5 in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi. d Schematic illustration showing the involvement of ALDH3A1, NDUFA1, or NDUFAF5 in lipid metabolic reprogramming and mitochondrial respiratory activity. e Relative OCR levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). f Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). ANOVA compared the difference in e and f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c , e and f

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Activity Assay, Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 facilitates tumorigenesis and aggressiveness of NB cells via interacting with ZRF1. a and b Representative images (left panel) and quantification (right panel) of soft agar (a) and matrigel invasion (b) assays indicating the growth and invasion of NB cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi. c Representative images (upper panel), in vivo growth curve, and weight at the end points (lower panel) of xenografts in nude mice formed by subcutaneous injection of SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi ( n = 5 for each group). d Representative images (upper panel) and quantification (lower panel) of immunohistochemical staining showing expression of Ki-67 and CD31 within xenografts ( n = 5 for each group). Scale bars: 50 μm. e and f Representative in vivo images (e), hematoxylin & eosin staining (f, upper panel), lung metastatic counts (f, lower panel), and Kaplan–Meier curves (f, lower panel) of nude mice treated with tail vein injection of SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi ( n = 5 for each group). Scale bars: 100 μm. ANOVA compared the difference in a - d and f . Log-rank test for survival comparison in f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in a and b

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, In Vivo, Injection, Immunohistochemical staining, Staining, Expressing

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: Therapeutic blocking p113-ZRF1 interaction inhibits NB progression. a 3D structure and sequences of inhibitory peptides (ZIP-12) for blocking interaction between p113 and ZRF1, and those of mutant control (Ctrl) peptides. b Confocal images showing the distribution of synthesized FITC-labeled Ctrl or ZIP-12 peptides (20 μmol·L − 1 , arrowheads) within cultured BE(2)-C cells, with nuclei and cellular membranes staining with DAPI or Dil. Scale bars: 10 μm. c Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. d Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. e In vivo images (left upper panel), growth curve (right panel), and weight at the end points (right panel) of xenografts formed by subcutaneous injection of BE(2)-C cells in nude mice ( n = 5 per group) that were treated with intravenous injection of Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (left lower panel). f In vivo imaging (left panel), lung metastatic colonization (right lower panel), and Kaplan–Meier curves (right lower panel) of nude mice ( n = 5 for each group) treated with tail vein injection of BE(2)-C cells, Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (right upper panel). Student’s t test or ANOVA compared the difference in d - f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01 vs. Ctrl. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b - d

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Blocking Assay, Mutagenesis, Synthesized, Labeling, Cell Culture, Staining, Co-Immunoprecipitation Assay, Western Blot, Activity Assay, In Vivo, Injection, In Vivo Imaging

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: CUX1 , ZRF1 , BRD4 and target genes are associated with poor outcome of NB patients. a Kaplan–Meier curves indicating overall survival of 498 well-defined NB cases (GSE62564) with high or low expression of CUX1 (cutoff value = 32.233), ZRF1 (cutoff value = 21.749), BRD4 (cutoff value = 652.58), ALDH3A1 (cutoff value = 1.181), NDUFA1 (cutoff value = 22.511), or NDUFAF5 (cutoff value = 7.964). b The positive expression correlation between ZRF1 and ALDH3A1 , NDUFA1 , or NDUFAF5 in 498 well-defined NB cases (GSE62564). c The mechanisms underlying p113-faciliated NB progression: as a novel protein encoded by ecircCUX1 , p113 cooperates with ZRF1 and BRD4 to activate the transcription of ALDH3A1 , NDUFA1 , or NDUFAF5 , resulting in promoted conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, mitochondrial complex I activity, growth, and aggressiveness of NB cells. Meanwhile, inhibitory peptides (ZIP-12) blocking p113-ZRF1 interaction suppresses tumor progression. Log-rank test for survival comparison in a . Pearson’s correlation coefficient for b

Article Snippet: ChIP assay was undertaken using EZ-ChIP kit (Upstate Biotechnology, Temacula, CA) [ – ], with antibodies specific for p113 (ABclonal Biotechnology Co., Ltd), ZRF1 (12844S), or BRD4 (13,440, Cell Signaling Technology Inc.) and primers (Additional file : Table S1).

Techniques: Expressing, Activity Assay, Blocking Assay