Journal: Signal Transduction and Targeted Therapy

Article Title: CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

doi: 10.1038/s41392-020-00375-5

Figure Lengend Snippet: CircRNA-SORE is overexpressed in sorafenib-resistant HCC cells. a Three sorafenib-resistant HCC cell lines (HepG2-SR, LM3-SR, and SKhep1-SR) were established and confirmed by the Real-time Cell Analysis xCELLigence System (Roche Applied Sciences). Curves demonstrate the inhibitory effect of sorafenib on parental (P) and sorafenib-resistant (SR) cell lines. b Hierarchical clustering of 14 upregulated circRNAs and 14 downregulated circRNAs in HepG2-SR cells compared with parental HepG2 cells by Arraystar Human CircRNA Array analysis. CircRNAs with fold change >2 and p < 0.05 were selected as significantly different. c Scatter plots (left panel) and volcano plots (right panel) of the differentially expressed circRNAs. Red dots indicate differentially expressed circRNAs ( p < 0.05 and fold change > 2). d CircRNA_104797 levels (normalized by GAPDH) in parental (P) and resistant (R) cell lines treated with the indicated concentrations of sorafenib (7 μM for HepG2, 6 μM for LM3 and SKhep1) for 72 h. e Diagram illustrating back-splicing of circRNA-SORE from the TLE4 host gene. f Time-course of the relative expression of circRNA-SORE and TLE4 in HepG2-SR cells treated with actinomycin D (10 µg/mL). g qPCR analysis of circRNA-SORE and GAPDH mRNA in HepG2-SR, LM3-SR and SKhep1-SR cells with or without RNase R treatment for 30 min at 37 °C. h Relative expression of circRNA-SORE in mRNA and non-mRNA samples in the indicated cell lines. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test. *** p < 0.001. NS not significant

Article Snippet: Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells.

Techniques: Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

doi: 10.1038/s41392-020-00375-5

Figure Lengend Snippet: CircRNA-SORE is critical for maintaining sorafenib resistance. a Relative cell viability of three HCC sorafenib-resistant cell lines with circRNA-SORE siRNA knockdown (si-circRNA-SORE) compared with controls treated with the indicated concentrations of sorafenib (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 72 h. b Colony formation assays in three HCC sorafenib-resistant cell lines with or without shcircRNA-SORE. Cells were all treated with sorafenib (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 24 h in complete media, washed with PBS and cultured in complete media for another 7 days. c HepG2-P and HepG2-SR cell lines transfected with si-circRNA-SORE or negative control siRNA (si-NC) were treated with sorafenib. Cell viability was recorded by Real-time Cell Analysis xCELLigence System and three technical repetitions were performed. ΔAUC (difference of area under curve) = (AUC NC) – (AUC si-SORE). Each technical repetition yields a ΔAUC, and t tests were performed. d Similar to ( c ) except no sorafenib was added. e Brightfield images (objective magnification = 10) showing cell morphology of three sorafenib-resistant cell lines with or without si-circRNA-SORE or sorafenib at the indicated concentrations (7 μM for HepG2-SR, 6 μM for LM3-SR and SKhep1-SR) for 72 h. Scale bar, 20 µm. f Western blot analysis of sorafenib-resistant cell lines transfected with si-circRNA-SORE using PARP antibody (upper) and β-tubulin antibody (lower). g PI/Annexin V flow cytometry analysis (10,000 cells) of sorafenib-resistant cells transfected with si-NC or si-circRNA-SORE. h TUNEL staining (green) of three sorafenib-resistant cell lines transfected with si-NC or si-circRNA-SORE. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. i Kaplan−Meier recurrence-free survival analysis for sorafenib-treated HCC patients with low and high circRNA-SORE expression. The median circRNA-SORE expression was used as cut-off for high- and low-expression groups. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test and Kaplan−Meier survival analysis. * p < 0.05, and ** p < 0.01

Article Snippet: Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells.

Techniques: Cell Culture, Transfection, Negative Control, Western Blot, Flow Cytometry, TUNEL Assay, Staining, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

doi: 10.1038/s41392-020-00375-5

Figure Lengend Snippet: CircRNA-SORE functions by binding to YBX1 protein. a Venn diagram illustrating the number of proteins identified using LC-MS/MS from circRNA-SORE pull-down samples in HepG2-SR cells (179 unique proteins, blue) compared with controls (949 unique proteins, red). The overlap (106 proteins, purple) indicates the number of proteins found in both samples. b Volcano plot illustrating the 129 upregulated and 301 downregulated proteins of interest (fold change > 2 and p < 0.05) in the si-circRNA-SORE group compared with controls in HepG2-SR cells. c Bubble diagram of KEGG pathway analysis in si-circRNA-SORE-dysregulated proteins shows the top 20 enriched pathways. d Biotinylated-probe pull-down assay for YBX1 using western blot and qPCR analysis for circRNA-SORE in HepG2-SR cells. The probe was designed according to the junction region of circRNA-SORE. e Reciprocal RNA immunoprecipitation using YBX1 for circRNA-SORE in HepG2-SR cells. f Biotinylated-probe pull-down assay for YBX1 in HepG2-SR cells with or without MAO treatment. The probe was designed according to the junction region of circRNA-SORE. g Reciprocal RNA immunoprecipitation using YBX1 for circRNA-SORE in HepG2-SR cells with or without MAO treatment. h Biotinylated-probe pull-down assay for YBX1 using western blot in HepG2-P cells overexpressing NC, circRNA-SORE and circRNA-SORE-M. circRNA-SORE-M: circRNA-SORE with a mutated YBX1-binding motif. The probe was designed according to the junction region of circRNA-SORE. i RNA immunoprecipitation using YBX1 for circRNA-SORE and circRNA-SORE-M in HepG2-P cells overexpressing circRNA-SORE and circRNA-SORE-M. j circRNA-SORE FISH (red), YBX1 immunofluorescence (green), nuclei staining (blue), and merged (yellow) images in HepG2-SR cells. Merged panel shows the colocalization of circRNA-SORE and YBX1. Scale bar, 20 µm. k Overall survival curve of HCC patients ( N = 364) from TCGA database according to high and low YBX1 expression (cut-off: median of YBX1 transcript per million; HR = 2). l Western blot analysis of YBX1 and β-tubulin in HCC sorafenib-resistant cells and parental cells. m Western blot analysis of YBX1, AKT, Raf1, ERK, c-Myc, TGF-β1 and β-tubulin in HepG2-SR cells transfected with si-NC or YBX1 siRNA. n Cell viability of sorafenib-resistant cells transfected with siRNA targeting YBX1 (si-YBX1-2). o PI/Annexin V staining and flow cytometry in sorafenib-resistant cells (10,000 cells) following si-NC or si-YBX1-2 transfection. p Western blot analysis of YBX1, AKT, Raf1, ERK, c-Myc, TGF-β1, and β-tubulin in sorafenib-resistant cells following si-circRNA-SORE transfection. q Western blot analysis of YBX1 and β-tubulin and cell viability in HepG2-SR cells transfected with circRNA-SORE siRNA and overexpressing (OE) YBX1 by lentivirus. r Western blot analysis of YBX1 and β-tubulin and cell viability in HepG2-P cells overexpressing (OE) circRNA-SORE by lentivirus and transfected with YBX1 siRNA. s , t Kaplan−Meier recurrence-free survival analysis and overall survival analysis for sorafenib-treated HCC patients according to low and high YBX1 protein levels. Cases were stratified as low (− and +) and high (++ and + ++) YBX1 protein expression by immunohistochemistry staining. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM. Statistical analyses used Student’s t test. * p < 0.05, ** p < 0.01 and *** p < 0.001. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test, Kaplan−Meier survival analysis, and log-rank test. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Pull Down Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Expressing, Transfection, Flow Cytometry, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

doi: 10.1038/s41392-020-00375-5

Figure Lengend Snippet: CircRNA-SORE transmits sorafenib resistance by exosomes. a Exosomes were extracted from the blood of nine HCC patients prior to sorafenib treatment, and exosomal circRNA-SORE expressions were evaluated by qPCR assays. Lower and higher circRNA-SORE expressions were stratified by the average expression among the nine patients. Response to sorafenib was defined as once achieved complete response, partial response or stable disease for >3 months. b Negative staining electron micrograph of exosomes isolated from HepG2-SR culture media. Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells. The chart shows the cell viability in exosome-treated parental cells. l BALB/c nude mice (4- to 6-week-old males) were subcutaneously injected with SKhep1-P cells. On week 4, the mice were treated with sorafenib (30 mg/kg/day) by oral gavage and exosomes (80 μg twice a week for 4 weeks) from SKhep1-SR or SKhep1-P cell culture media by injection at the implantation site twice a week for 4 weeks. Animals were sacrificed on week 8 and xenografts were isolated and measured. Three independent experiments with three technical repetitions were performed. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Article Snippet: Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells.

Techniques: Expressing, Negative Staining, Isolation, Western Blot, Marker, Cell Culture, Transfection, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

doi: 10.1038/s41392-020-00375-5

Figure Lengend Snippet: Silencing circRNA-SORE substantially improves sorafenib efficacy in vivo. a BALB/c nude mice (4- to 6-week-old males) were orthotopically implanted with SKhep1-SR-luc-NC or SKhep1-SR-luc-shcircRNA-SORE cells. On week 4, the mice were treated with sorafenib (30 mg/kg/day) for 4 weeks. In vivo luminescent imaging was performed weekly starting on week 4. Animals were sacrificed on week 8. Luminescence intensity ranges from low (blue) to high (red). b BALB/c nude mice (4- to 6-week-old males) were subcutaneously injected with LM3-SR cells. On week 4, the mice were treated with sorafenib (30 mg/kg/day) for 4 weeks and then injected at the implantation site with in vivo-grade si-circRNA-SORE or si-NC twice a week for 2 weeks. Animals were sacrificed on week 8. c NOD/SCID mice (4- to 6-week-old males) were subcutaneously implanted with HCC patient-derived xenograft (PDX) and treated with sorafenib (30 mg/kg/day) by oral gavage starting on week 4. Eight weeks later, the sorafenib-resistant xenograft was removed for a second round of orthotopic implantation. On week 4, the second PDX generation mice were treated with sorafenib (30 mg/kg/day) for 4 weeks and were injected at the implantation site with in vivo-grade si-circRNA-SORE or si-NC twice a week for 2 weeks. Animals were sacrificed and the second-generation PDX was isolated on week 8. d Immunohistochemistry for YBX1 in si-circRNA-SORE and si-NC LM3-SR xenografts. Scale bar, 50 µm. e Immunohistochemistry for YBX1 in si-circRNA-SORE and control PDX. Scale bar, 50 µm. f Model for circRNA-SORE-mediated sorafenib resistance in HCC. CircRNA-SORE is necessary for the development and maintenance of sorafenib resistance in HCC, and silencing circRNA-SORE substantially increases sorafenib-induced apoptosis. CircRNA-SORE specifically binds to the master oncogenic protein YBX1 and thus sequesters YBX1 in the cytoplasm, which prevents YBX1 ubiquitination and degradation by PRP19 in the nucleus. CircRNA-SORE is transmitted by exosomes, allowing for the spread of sorafenib resistance among HCC cells. Data are expressed as mean ± SEM (error bars). Statistical analyses used Student’s t test. *** p < 0.001

Article Snippet: Scale bar, 200 nm. c Size distribution of exosomes by NanoSight NS300 instrument. d Western blot analysis for positive exosome markers (HSP70, TSG101, CD9 and CD63) and negative exosome marker (Calnexin). e qPCR analysis of exosomal circRNA-SORE isolated from sorafenib-resistant and parental cell culture media. f qPCR analysis of circRNA-SORE isolated from sorafenib-resistant cell culture media (M) and cells (C). g Schematic showing that exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat (30 μg) parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. h , i Exosomes were isolated from parental and sorafenib-resistant cells (P-exo and SR-exo, respectively) and used to treat parental cells for 24 h. The exosome-treated parental cells were then transfected with si-NC or si-circRNA-SORE and treated with sorafenib for 48 h. Charts show h qPCR results of circRNA-SORE expression and i cell viability in exosome-treated parental cells. j Schematic showing that HepG2-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments (30 μg) on HepG2 parental cells. k HepG2-SR, SKhep1-SR and LM3-SR cells were treated with si-NC or si-circRNA-SORE before the culture media were harvested for exosome isolation and subsequent exosome treatments on parental cells.

Techniques: In Vivo, Imaging, Injection, Derivative Assay, Isolation, Immunohistochemistry