Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 physically interacts with MSI2 in PDAC cells. (A) Coomassie blue staining of cytoplasmic proteins pulled down by circPRKD3 antisense (AS) or sense (S) DNA probes in PANC-1 cells. (B) Representative MS/MS spectrum identifying the MSI2 peptide from MS analysis of the excised band. (C) RNA pull-down assays showing specific association of MSI2 with circPRKD3, as confirmed by immunoblot analysis (top) and RT-PCR detection of circRNA (bottom). PABPC1 and GAPDH proteins serve as nonspecific controls. (D) In vitro circularized sense strand (S) circPRKD3 specifically enriches endogenous MSI2 proteins from PANC-1 cell lysates. Probe loading was verified by streptavidin-HRP. (E) RIP assays demonstrating enrichment of circPRKD3 by anti-MSI2 antibody compared to IgG control. MYC mRNA and TUBA1B mRNA serve as positive and negative controls, respectively. (F) Schematic diagrams of MSI2 RNA-binding domains and truncated mutants. (G and H) Flag-RIP experiments were performed in PANC-1 cells expressing MSI2 truncated mutants. Expression and IP efficiency of MSI2 mutants were evaluated by immunoblot analysis using anti-Flag antibody (G). CircPRKD3 enrichment by MSI2 truncated mutants was measured by RT-qPCR analysis (H). (I and J) Flag-RIP assays conducted in PANC-1 cells expressing either WT MSI2 or RRM1 mutants. Immunoblot analysis with anti-Flag antibody confirmed expression and IP efficiency of MSI2 mutants (I). RT-qPCR quantification revealed reduced circPRKD3 enrichment by K22A or R100A mutants compared to WT MSI2 (J). MYC mRNA serves as positive controls. (K) Transwell migration assays demonstrated that K22A and R100A mutants impaired the pro-migratory effects of MSI2 in both PANC-1 and CFPAC-1 cells. ** P < 0.01.

Article Snippet: The following antibodies were used: Lamin A/C (Abways, #CY5222; 1:1,000), GAPDH (Cell Signaling Technology, #3683; 1:5,000), MSI2 (Proteintech, #10770; 1:1,000), PABPC1 (Proteintech, #10970; 1:1,000), Flag (Proteintech, #66008; 1:1,000), Tubulin (Abways, #AB0049; 1:5,000), Ubiquitin (Cell Signaling Technology, #3936; 1:1,000), β-TRCP (Cell Signaling Technology, #4394; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), N-cadherin (Cell Signaling Technology, #4061; 1:1,000), and Vimentin (Cell Signaling Technology, #3932; 1:1,000).

Techniques: Staining, Tandem Mass Spectroscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Control, RNA Binding Assay, Expressing, Quantitative RT-PCR, Migration

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 promotes PDAC metastasis through its interaction with MSI2 (A) Schematic diagram of 2 putative MSI2-binding regions (P1 and P2) containing the “UAGU” motifs (top) and sequences of WT (P1 and P2) and mutant (P1-mut and P2-mut) RNA probes (bottom). (B) RNA pull-down assays demonstrating MSI2 specifically binds to “UAGU”-containing P1/P2 probes in PANC-1 lysates. Both PABPC1 and tubulin serve as negative controls. Probe loading was verified by streptavidin-HRP. (C) RNA pull-down assays showing direct interaction between recombinant MSI2 proteins and biotinylated P1/P2 probes (but not mutants). (D) RNA-EMSAs confirming the direct binding of recombinant MSI2 protein with biotinylated P1/P2 probes. (E) RIP-qPCR showing MSI2 preferentially interacted with WT circPRKD3 (WT) over MSI2-binding-deficient mutants (Mut) in PDAC cells. (F and G) Transwell assays revealing that WT circPRKD3 but not MSI2-binding-deficient mutant (Mut) promoted migration and invasion of PANC-1 (F) and CFPAC-1 cells (G). Scale bar = 250 μm. (H and I) Lung metastasis model: NSIG mice ( n = 5 per group) intravenously injected with 1 × 10 6 luciferase-labeled CFPAC-1 cells expressing vector (Vector), WT circPRKD3, or MSI2-binding-deficient mutant (Mut). In vivo bioluminescence imaging and quantification of lung metastasis are shown in (H), and the representative H&E-stained lung sections showed metastatic nodules (I). Scale bar = 100 μm. T: tumor tissue; N: normal tissue. Arrows indicate metastatic lesions. ** P < 0.01.

Article Snippet: The following antibodies were used: Lamin A/C (Abways, #CY5222; 1:1,000), GAPDH (Cell Signaling Technology, #3683; 1:5,000), MSI2 (Proteintech, #10770; 1:1,000), PABPC1 (Proteintech, #10970; 1:1,000), Flag (Proteintech, #66008; 1:1,000), Tubulin (Abways, #AB0049; 1:5,000), Ubiquitin (Cell Signaling Technology, #3936; 1:1,000), β-TRCP (Cell Signaling Technology, #4394; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), N-cadherin (Cell Signaling Technology, #4061; 1:1,000), and Vimentin (Cell Signaling Technology, #3932; 1:1,000).

Techniques: Binding Assay, Mutagenesis, Recombinant, Migration, Injection, Luciferase, Labeling, Expressing, Plasmid Preparation, In Vivo, Imaging, Staining

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 stabilizes MSI2 by inhibiting its ubiquitin-proteasome degradation. (A and B) Immunoblot analysis showing that WT circPRKD3, but not its linear transcript mutant (Linear) or MSI2-binding-deficient mutant (Mut), up-regulated MSI2 protein levels in PANC-1 and CFPAC-1 cells. (C) CircPRKD3 depletion reduced endogenous MSI2 protein expression in PANC-1 and Capan-2 cells. (D and E) IHC staining of MSI2 protein in lung metastatic lesions from mice injected with CFPAC-1 cells expressing with vector, WT circPRKD3, linear transcript mutant (D), or MSI2-binding site mutant (Mut) (E). Scale bar = 100 μm. T: tumor; N: normal tissue. (F) Proteasome inhibition with MG132 (20 μM, 6 h) restored MSI2 protein levels in circPRK3-depleted cells. (G and H) Ubiquitination assays showing reduced MSI2 polyubiquitination levels in cells overexpressing WT circPRKD3 compared to mutants. (I) Enhanced MSI2 polyubiquitination following circPRKD3 knockdown.

Article Snippet: The following antibodies were used: Lamin A/C (Abways, #CY5222; 1:1,000), GAPDH (Cell Signaling Technology, #3683; 1:5,000), MSI2 (Proteintech, #10770; 1:1,000), PABPC1 (Proteintech, #10970; 1:1,000), Flag (Proteintech, #66008; 1:1,000), Tubulin (Abways, #AB0049; 1:5,000), Ubiquitin (Cell Signaling Technology, #3936; 1:1,000), β-TRCP (Cell Signaling Technology, #4394; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), N-cadherin (Cell Signaling Technology, #4061; 1:1,000), and Vimentin (Cell Signaling Technology, #3932; 1:1,000).

Techniques: Ubiquitin Proteomics, Western Blot, Mutagenesis, Binding Assay, Expressing, Immunohistochemistry, Injection, Plasmid Preparation, Inhibition, Knockdown

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 disrupts the interaction between MSI2 and β-TRCP. (A) Effects of β-TRCP knockdown on MSI2 expression in PANC-1 and CFPAC-1 cells. (B) Co-IP assays with anti-Flag antibody confirming physical interaction between Flag-MSI2 and β-TRCP in PANC-1 cells. (C) circPRKD3 overexpression reduced the MSI2–β-TRCP interaction in co-IP assays with anti-Flag antibody. (D) Co-IP assays with anti-MSI2 antibody showing that WT circPRKD3, but not MSI2-binding-deficient mutant (Mut), disrupted MSI2–β-TRCP complex formation in PANC-1 cells. (E and F) Co-IP assays showed that WT circPRKD3 decreased the association of β-TRCP with Flag-MSI2 (E) and endogenous MSI2 (F) compared with vector controls or linear transcript mutants (Linear). This inhibitory effect was abolished by RNase A treatment but remaining intact following RNase R digestion. (G) Depletion of circPRKD3 reduced MSI2 protein levels in PANC-1 cells, an effect that was rescued by concomitant β-TRCP knockdown.

Article Snippet: The following antibodies were used: Lamin A/C (Abways, #CY5222; 1:1,000), GAPDH (Cell Signaling Technology, #3683; 1:5,000), MSI2 (Proteintech, #10770; 1:1,000), PABPC1 (Proteintech, #10970; 1:1,000), Flag (Proteintech, #66008; 1:1,000), Tubulin (Abways, #AB0049; 1:5,000), Ubiquitin (Cell Signaling Technology, #3936; 1:1,000), β-TRCP (Cell Signaling Technology, #4394; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), N-cadherin (Cell Signaling Technology, #4061; 1:1,000), and Vimentin (Cell Signaling Technology, #3932; 1:1,000).

Techniques: Knockdown, Expressing, Co-Immunoprecipitation Assay, Over Expression, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: Clinical significance of the circPRKD3–MSI2 axis in PDAC. (A) MSI2 and circPRKD3 expression were measured by immunoblot (top) and RT-qPCR analysis (bottom) in paired PDAC tumors (C) and adjacent normal tissues (N). (B) Pearson correlation analysis between MSI2 protein and circPRKD3 levels in PDAC specimens. (C) Kaplan–Meier overall survival analysis of PDAC patients stratified by circPRKD3 expression. (D) RT-qPCR analysis of serum circPRKD3 levels in PDAC patients (Tumor) versus healthy controls (Normal) and patients with other pancreas-related diseases (Other diseases). (E and F) ROC analysis comparing the diagnostic performance of circPRKD3, conventional markers (CA19-9, CEA, and CA125), and combined panels for distinguishing PDAC from other pancreas-related diseases. (G) Diagnostic performance of circPRKD3/CA19-9 dual-marker panel in distinguishing PDAC patients from other pancreas-related diseases. (H) Proposed mechanism of circPRKD3-driven PDAC metastasis through MSI2 stabilization. * P < 0.05.

Article Snippet: The following antibodies were used: Lamin A/C (Abways, #CY5222; 1:1,000), GAPDH (Cell Signaling Technology, #3683; 1:5,000), MSI2 (Proteintech, #10770; 1:1,000), PABPC1 (Proteintech, #10970; 1:1,000), Flag (Proteintech, #66008; 1:1,000), Tubulin (Abways, #AB0049; 1:5,000), Ubiquitin (Cell Signaling Technology, #3936; 1:1,000), β-TRCP (Cell Signaling Technology, #4394; 1:1,000), E-cadherin (Cell Signaling Technology, #3195; 1:1,000), N-cadherin (Cell Signaling Technology, #4061; 1:1,000), and Vimentin (Cell Signaling Technology, #3932; 1:1,000).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Diagnostic Assay, Marker

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 physically interacts with MSI2 in PDAC cells. (A) Coomassie blue staining of cytoplasmic proteins pulled down by circPRKD3 antisense (AS) or sense (S) DNA probes in PANC-1 cells. (B) Representative MS/MS spectrum identifying the MSI2 peptide from MS analysis of the excised band. (C) RNA pull-down assays showing specific association of MSI2 with circPRKD3, as confirmed by immunoblot analysis (top) and RT-PCR detection of circRNA (bottom). PABPC1 and GAPDH proteins serve as nonspecific controls. (D) In vitro circularized sense strand (S) circPRKD3 specifically enriches endogenous MSI2 proteins from PANC-1 cell lysates. Probe loading was verified by streptavidin-HRP. (E) RIP assays demonstrating enrichment of circPRKD3 by anti-MSI2 antibody compared to IgG control. MYC mRNA and TUBA1B mRNA serve as positive and negative controls, respectively. (F) Schematic diagrams of MSI2 RNA-binding domains and truncated mutants. (G and H) Flag-RIP experiments were performed in PANC-1 cells expressing MSI2 truncated mutants. Expression and IP efficiency of MSI2 mutants were evaluated by immunoblot analysis using anti-Flag antibody (G). CircPRKD3 enrichment by MSI2 truncated mutants was measured by RT-qPCR analysis (H). (I and J) Flag-RIP assays conducted in PANC-1 cells expressing either WT MSI2 or RRM1 mutants. Immunoblot analysis with anti-Flag antibody confirmed expression and IP efficiency of MSI2 mutants (I). RT-qPCR quantification revealed reduced circPRKD3 enrichment by K22A or R100A mutants compared to WT MSI2 (J). MYC mRNA serves as positive controls. (K) Transwell migration assays demonstrated that K22A and R100A mutants impaired the pro-migratory effects of MSI2 in both PANC-1 and CFPAC-1 cells. ** P < 0.01.

Article Snippet: Following incubation overnight at 4 °C with MSI2 antibody (Proteintech, #10770, 1:400 dilution) or Nucleolin antibody (Abcam, #ab136649, 1:200 dilution), sections were treated with an HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Staining, Tandem Mass Spectroscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Control, RNA Binding Assay, Expressing, Quantitative RT-PCR, Migration

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 promotes PDAC metastasis through its interaction with MSI2 (A) Schematic diagram of 2 putative MSI2-binding regions (P1 and P2) containing the “UAGU” motifs (top) and sequences of WT (P1 and P2) and mutant (P1-mut and P2-mut) RNA probes (bottom). (B) RNA pull-down assays demonstrating MSI2 specifically binds to “UAGU”-containing P1/P2 probes in PANC-1 lysates. Both PABPC1 and tubulin serve as negative controls. Probe loading was verified by streptavidin-HRP. (C) RNA pull-down assays showing direct interaction between recombinant MSI2 proteins and biotinylated P1/P2 probes (but not mutants). (D) RNA-EMSAs confirming the direct binding of recombinant MSI2 protein with biotinylated P1/P2 probes. (E) RIP-qPCR showing MSI2 preferentially interacted with WT circPRKD3 (WT) over MSI2-binding-deficient mutants (Mut) in PDAC cells. (F and G) Transwell assays revealing that WT circPRKD3 but not MSI2-binding-deficient mutant (Mut) promoted migration and invasion of PANC-1 (F) and CFPAC-1 cells (G). Scale bar = 250 μm. (H and I) Lung metastasis model: NSIG mice ( n = 5 per group) intravenously injected with 1 × 10 6 luciferase-labeled CFPAC-1 cells expressing vector (Vector), WT circPRKD3, or MSI2-binding-deficient mutant (Mut). In vivo bioluminescence imaging and quantification of lung metastasis are shown in (H), and the representative H&E-stained lung sections showed metastatic nodules (I). Scale bar = 100 μm. T: tumor tissue; N: normal tissue. Arrows indicate metastatic lesions. ** P < 0.01.

Article Snippet: Following incubation overnight at 4 °C with MSI2 antibody (Proteintech, #10770, 1:400 dilution) or Nucleolin antibody (Abcam, #ab136649, 1:200 dilution), sections were treated with an HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Binding Assay, Mutagenesis, Recombinant, Migration, Injection, Luciferase, Labeling, Expressing, Plasmid Preparation, In Vivo, Imaging, Staining

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 stabilizes MSI2 by inhibiting its ubiquitin-proteasome degradation. (A and B) Immunoblot analysis showing that WT circPRKD3, but not its linear transcript mutant (Linear) or MSI2-binding-deficient mutant (Mut), up-regulated MSI2 protein levels in PANC-1 and CFPAC-1 cells. (C) CircPRKD3 depletion reduced endogenous MSI2 protein expression in PANC-1 and Capan-2 cells. (D and E) IHC staining of MSI2 protein in lung metastatic lesions from mice injected with CFPAC-1 cells expressing with vector, WT circPRKD3, linear transcript mutant (D), or MSI2-binding site mutant (Mut) (E). Scale bar = 100 μm. T: tumor; N: normal tissue. (F) Proteasome inhibition with MG132 (20 μM, 6 h) restored MSI2 protein levels in circPRK3-depleted cells. (G and H) Ubiquitination assays showing reduced MSI2 polyubiquitination levels in cells overexpressing WT circPRKD3 compared to mutants. (I) Enhanced MSI2 polyubiquitination following circPRKD3 knockdown.

Article Snippet: Following incubation overnight at 4 °C with MSI2 antibody (Proteintech, #10770, 1:400 dilution) or Nucleolin antibody (Abcam, #ab136649, 1:200 dilution), sections were treated with an HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Ubiquitin Proteomics, Western Blot, Mutagenesis, Binding Assay, Expressing, Immunohistochemistry, Injection, Plasmid Preparation, Inhibition, Knockdown

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 disrupts the interaction between MSI2 and β-TRCP. (A) Effects of β-TRCP knockdown on MSI2 expression in PANC-1 and CFPAC-1 cells. (B) Co-IP assays with anti-Flag antibody confirming physical interaction between Flag-MSI2 and β-TRCP in PANC-1 cells. (C) circPRKD3 overexpression reduced the MSI2–β-TRCP interaction in co-IP assays with anti-Flag antibody. (D) Co-IP assays with anti-MSI2 antibody showing that WT circPRKD3, but not MSI2-binding-deficient mutant (Mut), disrupted MSI2–β-TRCP complex formation in PANC-1 cells. (E and F) Co-IP assays showed that WT circPRKD3 decreased the association of β-TRCP with Flag-MSI2 (E) and endogenous MSI2 (F) compared with vector controls or linear transcript mutants (Linear). This inhibitory effect was abolished by RNase A treatment but remaining intact following RNase R digestion. (G) Depletion of circPRKD3 reduced MSI2 protein levels in PANC-1 cells, an effect that was rescued by concomitant β-TRCP knockdown.

Article Snippet: Following incubation overnight at 4 °C with MSI2 antibody (Proteintech, #10770, 1:400 dilution) or Nucleolin antibody (Abcam, #ab136649, 1:200 dilution), sections were treated with an HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Knockdown, Expressing, Co-Immunoprecipitation Assay, Over Expression, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: Clinical significance of the circPRKD3–MSI2 axis in PDAC. (A) MSI2 and circPRKD3 expression were measured by immunoblot (top) and RT-qPCR analysis (bottom) in paired PDAC tumors (C) and adjacent normal tissues (N). (B) Pearson correlation analysis between MSI2 protein and circPRKD3 levels in PDAC specimens. (C) Kaplan–Meier overall survival analysis of PDAC patients stratified by circPRKD3 expression. (D) RT-qPCR analysis of serum circPRKD3 levels in PDAC patients (Tumor) versus healthy controls (Normal) and patients with other pancreas-related diseases (Other diseases). (E and F) ROC analysis comparing the diagnostic performance of circPRKD3, conventional markers (CA19-9, CEA, and CA125), and combined panels for distinguishing PDAC from other pancreas-related diseases. (G) Diagnostic performance of circPRKD3/CA19-9 dual-marker panel in distinguishing PDAC patients from other pancreas-related diseases. (H) Proposed mechanism of circPRKD3-driven PDAC metastasis through MSI2 stabilization. * P < 0.05.

Article Snippet: Following incubation overnight at 4 °C with MSI2 antibody (Proteintech, #10770, 1:400 dilution) or Nucleolin antibody (Abcam, #ab136649, 1:200 dilution), sections were treated with an HRP-conjugated secondary antibody for 1 h at room temperature.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Diagnostic Assay, Marker

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 physically interacts with MSI2 in PDAC cells. (A) Coomassie blue staining of cytoplasmic proteins pulled down by circPRKD3 antisense (AS) or sense (S) DNA probes in PANC-1 cells. (B) Representative MS/MS spectrum identifying the MSI2 peptide from MS analysis of the excised band. (C) RNA pull-down assays showing specific association of MSI2 with circPRKD3, as confirmed by immunoblot analysis (top) and RT-PCR detection of circRNA (bottom). PABPC1 and GAPDH proteins serve as nonspecific controls. (D) In vitro circularized sense strand (S) circPRKD3 specifically enriches endogenous MSI2 proteins from PANC-1 cell lysates. Probe loading was verified by streptavidin-HRP. (E) RIP assays demonstrating enrichment of circPRKD3 by anti-MSI2 antibody compared to IgG control. MYC mRNA and TUBA1B mRNA serve as positive and negative controls, respectively. (F) Schematic diagrams of MSI2 RNA-binding domains and truncated mutants. (G and H) Flag-RIP experiments were performed in PANC-1 cells expressing MSI2 truncated mutants. Expression and IP efficiency of MSI2 mutants were evaluated by immunoblot analysis using anti-Flag antibody (G). CircPRKD3 enrichment by MSI2 truncated mutants was measured by RT-qPCR analysis (H). (I and J) Flag-RIP assays conducted in PANC-1 cells expressing either WT MSI2 or RRM1 mutants. Immunoblot analysis with anti-Flag antibody confirmed expression and IP efficiency of MSI2 mutants (I). RT-qPCR quantification revealed reduced circPRKD3 enrichment by K22A or R100A mutants compared to WT MSI2 (J). MYC mRNA serves as positive controls. (K) Transwell migration assays demonstrated that K22A and R100A mutants impaired the pro-migratory effects of MSI2 in both PANC-1 and CFPAC-1 cells. ** P < 0.01.

Article Snippet: After centrifugation at 16,000 × g for 15 min at 4 °C, the resulting supernatants were immunoprecipitated with 2 μg of anti-MSI2 antibody (Proteintech, #10770) or control IgG overnight at 4 °C with rotation.

Techniques: Staining, Tandem Mass Spectroscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, Control, RNA Binding Assay, Expressing, Quantitative RT-PCR, Migration

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 promotes PDAC metastasis through its interaction with MSI2 (A) Schematic diagram of 2 putative MSI2-binding regions (P1 and P2) containing the “UAGU” motifs (top) and sequences of WT (P1 and P2) and mutant (P1-mut and P2-mut) RNA probes (bottom). (B) RNA pull-down assays demonstrating MSI2 specifically binds to “UAGU”-containing P1/P2 probes in PANC-1 lysates. Both PABPC1 and tubulin serve as negative controls. Probe loading was verified by streptavidin-HRP. (C) RNA pull-down assays showing direct interaction between recombinant MSI2 proteins and biotinylated P1/P2 probes (but not mutants). (D) RNA-EMSAs confirming the direct binding of recombinant MSI2 protein with biotinylated P1/P2 probes. (E) RIP-qPCR showing MSI2 preferentially interacted with WT circPRKD3 (WT) over MSI2-binding-deficient mutants (Mut) in PDAC cells. (F and G) Transwell assays revealing that WT circPRKD3 but not MSI2-binding-deficient mutant (Mut) promoted migration and invasion of PANC-1 (F) and CFPAC-1 cells (G). Scale bar = 250 μm. (H and I) Lung metastasis model: NSIG mice ( n = 5 per group) intravenously injected with 1 × 10 6 luciferase-labeled CFPAC-1 cells expressing vector (Vector), WT circPRKD3, or MSI2-binding-deficient mutant (Mut). In vivo bioluminescence imaging and quantification of lung metastasis are shown in (H), and the representative H&E-stained lung sections showed metastatic nodules (I). Scale bar = 100 μm. T: tumor tissue; N: normal tissue. Arrows indicate metastatic lesions. ** P < 0.01.

Article Snippet: After centrifugation at 16,000 × g for 15 min at 4 °C, the resulting supernatants were immunoprecipitated with 2 μg of anti-MSI2 antibody (Proteintech, #10770) or control IgG overnight at 4 °C with rotation.

Techniques: Binding Assay, Mutagenesis, Recombinant, Migration, Injection, Luciferase, Labeling, Expressing, Plasmid Preparation, In Vivo, Imaging, Staining

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 stabilizes MSI2 by inhibiting its ubiquitin-proteasome degradation. (A and B) Immunoblot analysis showing that WT circPRKD3, but not its linear transcript mutant (Linear) or MSI2-binding-deficient mutant (Mut), up-regulated MSI2 protein levels in PANC-1 and CFPAC-1 cells. (C) CircPRKD3 depletion reduced endogenous MSI2 protein expression in PANC-1 and Capan-2 cells. (D and E) IHC staining of MSI2 protein in lung metastatic lesions from mice injected with CFPAC-1 cells expressing with vector, WT circPRKD3, linear transcript mutant (D), or MSI2-binding site mutant (Mut) (E). Scale bar = 100 μm. T: tumor; N: normal tissue. (F) Proteasome inhibition with MG132 (20 μM, 6 h) restored MSI2 protein levels in circPRK3-depleted cells. (G and H) Ubiquitination assays showing reduced MSI2 polyubiquitination levels in cells overexpressing WT circPRKD3 compared to mutants. (I) Enhanced MSI2 polyubiquitination following circPRKD3 knockdown.

Article Snippet: After centrifugation at 16,000 × g for 15 min at 4 °C, the resulting supernatants were immunoprecipitated with 2 μg of anti-MSI2 antibody (Proteintech, #10770) or control IgG overnight at 4 °C with rotation.

Techniques: Ubiquitin Proteomics, Western Blot, Mutagenesis, Binding Assay, Expressing, Immunohistochemistry, Injection, Plasmid Preparation, Inhibition, Knockdown

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: CircPRKD3 disrupts the interaction between MSI2 and β-TRCP. (A) Effects of β-TRCP knockdown on MSI2 expression in PANC-1 and CFPAC-1 cells. (B) Co-IP assays with anti-Flag antibody confirming physical interaction between Flag-MSI2 and β-TRCP in PANC-1 cells. (C) circPRKD3 overexpression reduced the MSI2–β-TRCP interaction in co-IP assays with anti-Flag antibody. (D) Co-IP assays with anti-MSI2 antibody showing that WT circPRKD3, but not MSI2-binding-deficient mutant (Mut), disrupted MSI2–β-TRCP complex formation in PANC-1 cells. (E and F) Co-IP assays showed that WT circPRKD3 decreased the association of β-TRCP with Flag-MSI2 (E) and endogenous MSI2 (F) compared with vector controls or linear transcript mutants (Linear). This inhibitory effect was abolished by RNase A treatment but remaining intact following RNase R digestion. (G) Depletion of circPRKD3 reduced MSI2 protein levels in PANC-1 cells, an effect that was rescued by concomitant β-TRCP knockdown.

Article Snippet: After centrifugation at 16,000 × g for 15 min at 4 °C, the resulting supernatants were immunoprecipitated with 2 μg of anti-MSI2 antibody (Proteintech, #10770) or control IgG overnight at 4 °C with rotation.

Techniques: Knockdown, Expressing, Co-Immunoprecipitation Assay, Over Expression, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: Research

Article Title: A Circular RNA Promotes Tumor Metastasis through Stabilizing MSI2 Protein in Pancreatic Ductal Adenocarcinoma

doi: 10.34133/research.0918

Figure Lengend Snippet: Clinical significance of the circPRKD3–MSI2 axis in PDAC. (A) MSI2 and circPRKD3 expression were measured by immunoblot (top) and RT-qPCR analysis (bottom) in paired PDAC tumors (C) and adjacent normal tissues (N). (B) Pearson correlation analysis between MSI2 protein and circPRKD3 levels in PDAC specimens. (C) Kaplan–Meier overall survival analysis of PDAC patients stratified by circPRKD3 expression. (D) RT-qPCR analysis of serum circPRKD3 levels in PDAC patients (Tumor) versus healthy controls (Normal) and patients with other pancreas-related diseases (Other diseases). (E and F) ROC analysis comparing the diagnostic performance of circPRKD3, conventional markers (CA19-9, CEA, and CA125), and combined panels for distinguishing PDAC from other pancreas-related diseases. (G) Diagnostic performance of circPRKD3/CA19-9 dual-marker panel in distinguishing PDAC patients from other pancreas-related diseases. (H) Proposed mechanism of circPRKD3-driven PDAC metastasis through MSI2 stabilization. * P < 0.05.

Article Snippet: After centrifugation at 16,000 × g for 15 min at 4 °C, the resulting supernatants were immunoprecipitated with 2 μg of anti-MSI2 antibody (Proteintech, #10770) or control IgG overnight at 4 °C with rotation.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Diagnostic Assay, Marker