Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: m 6 A‐modified circTACC3 is up‐regulated in MASH‐related HCC. (A) Schematic representation of m 6 A‐circRNA epitranscriptomic microarray assay. (B) Hierarchical clustering heatmap of differentially m 6 A modified circRNAs from MASH‐related HCC tumor and paired peritumoral normal tissues (m 6 A‐circRNA epitranscriptomic microarray assay; absolute m 6 A modification quantity shown, n = 5). (C) Venn diagram of overlapping circRNAs in MASH‐related HCC tumors with concurrent increases in absolute m 6 A modification quantity, relative m 6 A modification rate, and relative expression levels. (D‐E) The circTACC3 level in MASLD tissues, MASH‐related HCC tumor tissues, and paired peritumoral normal tissues ( n = 62) determined by ISH assay (D) and corresponding expression score analysis (E). ** P < 0.01; *** P < 0.001; NS , not significant. (F) MeRIP assay shows the enrichment of m 6 A‐modified circTACC3 in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues ( n = 3). (G) 3D‐FISH performed on MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids. Left upper panel shows circTACC3 (red) and DAPI (blue). Left lower panel shows the merge in 3D view. Right upper panel shows representative z‐stack layer capture. Right lower panel shows depth coding. (H) Nuclear‐cytoplasmic fractionation assay determined circTACC3 expression in nuclear and cytoplasmic fractionation, respectively ( n = 4). ** P < 0.01; *** P < 0.001; NS , not significant. (I) Schematic representation of exon 4 back‐splicing, circTACC3 forming, and the design of indicated primers. (J) Electrophoresis of RT‐PCR product amplified from cDNA or gDNA. (K) The expression of circTACC3 and TACC3 homologous mRNA from RNA with or without RNase R treatment. (L) Levels of circTACC3 and TACC3 mRNA in indicated cells that were treated with or without actinomycin D ( n = 4). *** P < 0.001. Abbreviations: m 6 A, N6‐methyladenosine; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; ISH, in situ hybridization‐ immunofluorescence; MeRIP, methylated RNA immunoprecipitation; 3D, three dimensions; FISH, fluorescence in situ hybridization; NAS, non‐alcoholic fatty liver disease activity score; RT‐PCR, reverse transcription‐PCR.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Microarray, Expressing, In Situ Hybridization, Derivative Assay, Fractionation, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Methylation, RNA Immunoprecipitation, Fluorescence, Activity Assay, Reverse Transcription

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3 m 6 A modification is associated with its intracellular localization. (A) Representative image of H&E staining of MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids with relatively low (#1) and high (#2) circTACC3 expression, treated with or without PA + OA. (B) 3D fluorescence scanning of 3‐OH BrDU probe (green), anti‐Ki‐67 antibody (magenta), and DAPI (blue) indicated tissue derived organoids, treated with or without PA + OA; maximum projection of signals is shown. (C‐D) Assays of fluorescence staining with the 3‐OH BrDU probe were conducted on the cell lines with different nuclear circTACC3 level (C, n = 6) and circTACC3 knockout cell strains or negative controls (D, n = 9). The numbers on the Y‐axis represent the apoptotic rate normalized to baseline of 1. ** P < 0.01; *** P < 0.001; NS , not significant. (E) MeRIP assay shows an enrichment of m 6 A‐modified circTACC3 in cytoplasmic and nuclear fraction of HCCLM3 cells. (F) Representation of predicted m 6 A modification motif of circTACC3 (predicted using the SRAMP website). (G) Absolute quantitative RT‐qPCR of m 6 A RNA shows m 6 A modification ratio of intra‐nuclear circTACC3 in the individual motif scale. (H) The m 6 A modification levels of circTACC3 were evaluated by MeRIP in m 6 A inhibitor treated group compared to control (DMSO) group, and in PA + OA treated group compared to control (Mock) group, respectively ( n = 3). *** P < 0.001. (I) Nucleo‐plasmic fractionation to evaluate circTACC3 expression after indicated treatment ( n = 4). *** P < 0.001. (J) 3D‐FISH conducted on MASH‐related HCC tumor tissue derived organoids demonstrates the 3D distribution of circTACC3 (red) in nuclei (blue). (K) H&E staining of MASH‐related HCC tumor tissue derived organoids treated with PA + OA and/or m 6 A intervention. Abbreviations: PA, palmitic acid; OA, oleic acid; T, tumor tissues; PT, peritumoral normal tissue; H&E, hematoxylin and eosin; MeRIP, methylated RNA immunoprecipitation; m 6 A, N6‐methyladenosine; NAS, Non‐alcoholic fatty liver disease activity score; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; FISH, fluorescence i n situ hybridization.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Staining, Derivative Assay, Expressing, Fluorescence, Knock-Out, Quantitative RT-PCR, Control, Fractionation, Methylation, RNA Immunoprecipitation, Activity Assay, Hybridization

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: The m 6 A modification of circTACC3 regulates its interaction with NONO/p54 nrb . (A) Western blot validation of NONO/p54 nrb pulldown by F2‐circTACC3 in HCCLM3 cells. (B) RIP assay shows enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA in HCCLM3 cells ( n = 4). *** P < 0.001. (C) FLIM‐FRET assay (left panel) and the schematic diagram of FLIM‐FRET (right panel) in PA and OA treated HCCLM3 and HepG2 cells. (D) CLIP assay followed by RT‐PCR gel electrophoresis in HCCLM3 cells. (E) The location of circTACC3 (red), NONO/p54 nrb (green), and LncNEAT1 (magenta) in nuclei (blue) was evaluated in HCCLM3 and HepG2 cells following PA + OA treatment. (F) The distribution of circTACC3 (red) in nuclei (blue) was assessed following NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. (G) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 following NONO/p54 nrb and LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (H) RIP assay shows the enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA following indicated treatment ( n = 3) in HCCLM3 and HepG2 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (I‐K) Representative fluorescence images show the distribution of circTACC3 (red) and NONO/p54 nrb (green) in nuclei (blue) in HCCLM3, HepG2 cells, and Hep3B cells expressed ectopic circTACC3 following indicated treatment. (L) RIP assay shows enrichment of wildtype/m 6 A modification site mutant circTACC3 in NONO/p54 nrb ‐associated RNA in PA and OA treated HCCLM3 and HepG2 cells. Data normalization by dividing anti‐NONO/p54 nrb RIP and IgG RIP values by their respective Input group data ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. Abbreviations: NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; CLIP, ultraviolet cross‐linking immunoprecipitation; RT‐PCR, reverse transcription‐PCR; FLIM‐FRET, fluorescence lifetime imaging microscopy‐forster resonance energy transfer; MFD, Minimal Fraction of Donor; PA, palmitic acid; OA, oleic acid; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; RIP, RNA immunoprecipitation.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Western Blot, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Fractionation, Fluorescence, Mutagenesis, Binding Assay, Cross-linking Immunoprecipitation, Reverse Transcription, Imaging, Microscopy, Förster Resonance Energy Transfer, Activity Assay, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop structure formation in the MASH‐related HCC genome. (A) Schematic representation of procedure to identify circR loops and circTACC3‐R loops. (B) Dot‐blot assay to validate the R loop structure. S9.6 antibody was used to specifically recognize DNA‐RNA hybrids. (C) DRIP assay to identify circTACC3 enrichment in R loop structures in HCCLM3 cells ( n = 4). ** P < 0.01; *** P < 0.001; ND , not detection. (D) Representative fluorescence images show the expression and aggregation of circTACC3 and R loops as glow and spectrum signal intensities in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues, respectively. (E) The 3D distribution of circTACC3 (red) and R loops (green) were showed by 3D‐FISH‐IF in tissue derived organoids. (F‐I) Representative fluorescence images (F, H) and peak graphs of the linear ROI (region of interest) (G, I) show the location of the indicated molecules in HCC cells following PA + OA treatment. The linear ROI is represented by a solid line in the fluorescence graph. (J) The localization of circTACC3 (red) and S9.6‐stained R loops (green) were validated after NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. Abbreviations: PA, palmitic acid; OA, oleic acid; DRIP, DNA‐RNA immunoprecipitation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Dot Blot, Fluorescence, Expressing, Derivative Assay, Staining, RNA Immunoprecipitation, Immunofluorescence, Hybridization, Activity Assay, Binding Assay, Mutagenesis

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop formation is regulated by lipid overload and m 6 A modification. (A) 3D‐distribution of circTACC3 (glow) and S9.6‐stained R loops (spectrum) in nuclei (blue) of MASH‐HCC tissue derived organoids after indicated treatment. (B‐C) DRIP assay shows the enrichment of circTACC3 in R loop structure following m 6 A modification interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 3). * P < 0.05; ** P < 0.01. (D) Representative fluorescence images show S9.6‐stained R loops (green) in PA + OA induced HCC cells transfected with the RNase H1‐Tet‐On system after treatment with or without Dox. (E) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (F‐G) Representative fluorescence pictures (F) and peak graphs of the linear ROI (G) demonstrating the colocalization of circTACC3 (red), S9.6‐indicated R loops (green), and NONO/p54 nrb (yellow) in nuclei (blue) with or without Dox‐inducible RNase H1 expression in PA and OA treated HCCLM3 and HepG2 cells. Abbreviations: MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; PA, palmitic acid; OA, oleic acid; T, tumor tissue; PT, peritumoral normal tissue; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; DRIP, DNA‐RNA immunoprecipitation; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Staining, Derivative Assay, Fluorescence, Transfection, Fractionation, Expressing, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loops aggregated to promote the inter‐TADs contact. (A) After DRIP‐ChIRP sequencing, the reads distributions across peaks of all independent biological replicates are presented. (B) Genome‐wide distribution of the circTACC3‐R Loop‐located genes positively correlated with PA + OA induction or negatively correlated with m 6 A modification intervention. (C) Representative fluorescence images of the colocalization of indicated molecules in nuclei of HCC cells treated with or without PA and OA following RNase R treatment. (D) Schematic representation shows combination of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq to analyze the distribution of the DSB‐circTACC3‐R Loop structures in the genome. (E) Genome‐wide distribution of the DSB‐circTACC3‐R loop located genes in PA + OA induced HepG2 cells. (F) Top four enriched DSB‐circTACC3‐R loop‐binding motifs based on de novo motif analysis. (G) The dynamic clustering of paraspeckles (indicated by NONO/p54 nrb ‐mCherry) were filmed using STELLARIS Dynamic Signal Enhancement 24 h after PA + OA induction at 5‐min intervals for a duration of 1.5 h. Examples (from 50 min to 85 min) of fusions of several NONO/p54 nrb ‐mCherry foci are shown (time points indicated in minutes). (H) Heatmap depicting the fold change(log 2 ) in Hi‐C contact frequencies between PA + OA‐treated and control cells throughout chromosome 7. Interactions that increase in PA + OA group (red) or decrease in Mock group (blue) are evident. Profile of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq are shown on the top. TADs that had higher inter‐TADs contact frequencies (named “contact‐elevated TADs”) in both long‐range (green box) and between adjacent TADs (red box) are marked. DSB‐circTACC3‐R loops are marked with red arrow. (I) Hi‐C maps around the human STX6 locus that formed DSB‐circTACC3‐R loop structure are shown. DSB‐circTACC3‐R loops are marked with red arrow. Abbreviations: DRIP, DNA‐RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; PA, palmitic acid; OA, oleic acid; Hi‐C, high‐throughput/resolution chromosome conformation capture; DSB, DNA double‐strand breaks; STX6, Syntaxin 6.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Sequencing, Genome Wide, Modification, Fluorescence, Binding Assay, Hi-C, Control, RNA Immunoprecipitation, Isolation, Purification, Residue, Variant Assay, Immunofluorescence, Hybridization, High Throughput Screening Assay

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loop‐localized genes are selectively activated. (A) List of DSB‐circTACC3‐R loop‐localized genes. (B) DSB‐circTACC3‐loop‐localized genes expression in HepG2 cells with/without lipid overload induction ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (C‐D) DRIP‐ChIRP‐seq (C) and γH2AX CUT&Tag‐seq (D) RPKM analysis of DSB‐circTACC3‐loop‐localized genes to compare circTACC3‐R Loop enrichment within the “contact‐elevated TADs” ( n = 26) or not within the “contact‐elevated TADs” ( n = 12). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (E‐F) Representative fluorescence images (E) and peaks graphs of the linear ROI (F) show the colocalization of indicated molecules with or without Dox‐inducible RNase H1 expression. (G) NONO/p54 nrb ‐mCherry HepG2 cells with or without Dox‐inducible RNase H1 expression were filmed 24 h after PA + OA induction at 5‐min intervals. Abbreviations: PA, palmitic acid; OA, oleic acid; TAD, topologically associated domain; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; RPKM, reads per kilobase per million mapped reads; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Expressing, Fluorescence, Residue, Variant Assay