Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 regulates the phenotypic plasticity of CAFs and promote PDAC progression by IL6. A Gene Set Enrichment Analysis (GSEA) of affected signatures in fibroblasts (circCUL2 vs. control). B GSEA plots for inflammatory CAF (iCAF) signatures in circCUL2 overexpression NFs from control. C - D qRT–PCR analysis of iCAF markers (IL6, TNF-α and IL1α) and myCAF marker (Acta2 and Axin2) expression in circCUL2 overexpression NFs. E Flow cytometric analysis of PDGFRα and α-SMA expression in circCUL2 overexpression NFs. F - H . Representative cytokine arrays for circCUL2-overexpression NFs and control (n = 3). arrows indicate the cytokines with significant changes, which were further confirmed by qRT–PCR and ELISA. I - L EdU assay ( I ), colony formation ( J ), Scratch wound healing assays ( K ) and transwell assays ( L ) of PANC-1 cells treated with conditioned medium circCUL2-overexpression NFs or anti-IL6. Scale bar, 100 μm. M western blot analysis of STAT3 and p-STAT3 in PANC-1 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, Quantitative RT-PCR, Marker, Expressing, Enzyme-linked Immunosorbent Assay, EdU Assay, Western Blot, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2-overexpression NFs promote PDAC progression in vivo. A Representative Bioluminescence images, lung and HE staining of lung tissue of mice 4 weeks after tail vein injection of luc-PANC-1 cell treated with conditioned medium as indicated (n = 8 per group). Scale bar, 100 μm. B Relative luminescence intensity in each group. C Histogram analysis of the metastatic nodules number in per lung. D lung metastasis rate of each group (Chi-square test). E - F Representative bioluminescence images and histogram analysis of luminescence intensity in each at day 30 are shown (n = 6). G Abdominal metastasis rate was calculated for indicated group (Chi-square test). H Representative images of orthotopic model in each group on which autopsy was performed. Red arrow indicated primary tumor; S, spleen; T, primary tumor; M, metastasis. I Images of PDX from 2 patients in 5 mice. ( J ) Tumor growth curves of indicated group (n = 5). K qRT–PCR analysis of circCUL2 levels in PDX of mice before and after treatment. L Representative images of IHC for PDGFRα and IL6. Scale bar, 100 μm. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, In Vivo, Staining, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: MyD88 is a direct downstream target gene of miR-203a-3p. A - B Colony formation and transwell assays of PANC-1 cells treated with conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. Scale bar: 100 μm. C - D ELISA assays detected IL6 level of conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. E Venn analysis of the potential downstream target genes of miR-203a-3p, predicted by miRTarbase, miRWalk and Tarbase. F - G qRT–PCR analysis of screened downstream target genes of miR-203a-3p in indicated NFs and CAFs. H Luciferase reporter assay was used to detect the luciferase activity of MyD88-Wild Type (MyD88-wt) and miR-203a-3p binding site mutated MyD88 (mYD88-mut) luciferase reporter cotransfected with miR-203a-3p mimic. N.S., no significant. I - J Western blotting analysis of MyD88, p65, pp65, IKBα and p-IKBα expression after overexpression of circCUL2 or silence of miR-203a-3p in NFs, or silence of circCUL2 or overexpression of miR-203a-3p in CAFs. GAPDH as a loading control. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 promotes proliferation and migration via MyD88/NF-κB/IL6 axis. A - D Cotransfection of circCUL2 overexpression plasmid and miR-203a-3p mimic in NFs or circCUL2 siRNA and miR-203-3p inhibitor in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα, and secretion level of IL6. E - F PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with miR-203a-3p mimic, or CAFs cotransfected circCUL2 siRNA and miR-203-3p inhibitor for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. G - J Cotransfection of circCUL2 overexpression plasmid and MyD88 siRNA in NFs or circCUL2 siRNA and MyD88 overexpression plasmid in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα and secretion level of IL6. K - L PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with MyD88 siRNA, or CAFs cotransfected circCUL2 siRNA and MyD88 overexpression plasmid for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Migration, Cotransfection, Over Expression, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: Clinical implication of circCUL2/miR-203a-3p/IL6 axis in PDAC. A , F , K The relative expression of miR-203a-3p ( A ), MyD88 ( F ) and IL6 ( G ) in 161 PDAC tissues compared to paired NAT. B - C , G - H , L - M Association analysis between miR-203a-3p ( B - C ), MyD88 ( G - H ) and IL6 ( L - M ) expression levels and LN status and tumor stages in 161 PDAC tissues. ( D - E , I - J , N - O ) Kaplan-Meier analysis of the correlation between miR-203a-3p ( D - E ), MyD88 ( I - J ) and IL6 ( N - O ) expression levels and OS or DFS of 161 PDAC patients. The median miR-203a-3p, MyD88 and IL6 expression was used as the cutoff value. P Proposed model indicates the mechanism by which circCUL2 activated iCAF phenotype and production of IL6 to promote malignant progression of PDAC via miR-203a-3p/MyD88/NF-κB pathway. Data are expressed as the mean ± SD. * p < 0.05 and *** p < 0.001

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Expressing