Journal: Heliyon

Article Title: MiRNA-20b/SUFU/Wnt axis accelerates gastric cancer cell proliferation, migration and EMT

doi: 10.1016/j.heliyon.2021.e06695

Figure Lengend Snippet: Overexpression of miRNA-20b induces Wnt pathway activation and EMT. (A)The expression of miRNA-20b in GC cell lines. (B) The transfection efficacy of miRNA-20b mimic and inhibitor. (C) Inhibition of miRNA-20b repressed β-catenin dependent luciferase activity. Topflash/Fopflash plasmid, pTK-renilla vector and miRNA-20b inhibitors were cotransfected into BGC823 cells. Top/Fopflash activity was calculated. T test was conducted. P = 0.014. (D) β-catenin nuclear translocation is regulated by miRNA-20b. MiRNA-20b inhibitors or mimics were transfected into BGC823 cells or HFE145 cells with respectively. Nuclear and cytoplasmic cell lystes were extracted. Immunoblotting of β-catenin was performed. (E) Inhibition of miRNA-20b blocked β-catenin nuclear translocation. MiRNA-20b inhibitors were transfected into BCG-823 cells. Confocal fluorescence of β-catenin was detected to reveal the β-catenin distribution. (F) MiRNA-20b modulated the expression level of Wnt pathway target genes. MiRNA-20-inhibitors or mimics were transfected into BGC823 cells or HFE145 cells respectively. The expression level of Wnt signaling downstream genes was determined by immunoblotting. (G) MiRNA-20b induced EMT. MiRNA-20 inhibitors or mimics were transfected into BGC823 cells or HFE145 cells with respectively. EMT markers were blotted. Each experiment was repeated three times.

Article Snippet: Antibodies we used include SUFU(Cell Signaling, (C81H7)#2522), Wnt/β-Catenin Activated Targets Antibody Sampler Kit (Cell Signaling,#8655), including CD44 (156-3C11),#3570; Cyclin D1 (92G2),#2978; c-Jun (60A8),#9165; LEF1 (C12A5),#2230; Met (D1C2),#8198; MMP-7 (D4H5),#3801; c-Myc (D84C12),#5605; TCF1/TCF7(C63D9),#2203; Anti-mouse IgG (#7076); Anti-rabbit IgG (#7074) and Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (1:1000, Cell Signaling,#9782) including Vimentin (D21H3) #5741; N-Cadherin (D4R1H)#13116; Claudin-1 (D5H1D)#13255; β-Catenin (D10A8) #8480; ZO-1 (D7D12)#8193; Snail (C15D3)#3879; Slug (C19G7)#9585; ZEB1 (D80D3)#3396; E-Cadherin (24E10) #3195; Anti-rabbit IgG#7074 and GAPDH antibody (1:5000, Cell Signaling, (D16H11)#5174S).

Techniques: Over Expression, Activation Assay, Expressing, Transfection, Inhibition, Luciferase, Activity Assay, Plasmid Preparation, Translocation Assay, Western Blot, Fluorescence

Journal: Heliyon

Article Title: MiRNA-20b/SUFU/Wnt axis accelerates gastric cancer cell proliferation, migration and EMT

doi: 10.1016/j.heliyon.2021.e06695

Figure Lengend Snippet: MiRNA-20b/SUFU/Wnt axis induces EMT. (A–E) MiRNA-20b inhibitors or miRNA-20b inhibitors combined with SUFU siRNAs were transfected into BGC823 cells. (A) Suppression of miRNA-20b inhibited β-catenin dependent luciferase activity and knockdown of SUFU partially alleviated the inhibition. NSC vs 20-inh, P = 0.000; 20-inh vs 20-inh + SUFU si, P = 0.021. (B–C) Inhibition of β-catenin nuclear translocation caused by miRNA-20b repression was relieved by reduction of SUFU. Western blotting (B) and confocal immunofluorescence (C) of β-catenin was performed to reveal the β-catenin cytoplasmic or nuclear localization. (D) Wnt signaling inhibition caused by miRNA-20b suppression was rescued by coupled transfection of SUFU siRNAs. The cell lysates were immunoblotted with antibodies of Wnt signaling downstream genes. (E) MiRNA-20b/SUFU/Wnt axis induces EMT. The cell lysates were blotted with antibodies of EMT markers. Each experiment was repeated three times.

Article Snippet: Antibodies we used include SUFU(Cell Signaling, (C81H7)#2522), Wnt/β-Catenin Activated Targets Antibody Sampler Kit (Cell Signaling,#8655), including CD44 (156-3C11),#3570; Cyclin D1 (92G2),#2978; c-Jun (60A8),#9165; LEF1 (C12A5),#2230; Met (D1C2),#8198; MMP-7 (D4H5),#3801; c-Myc (D84C12),#5605; TCF1/TCF7(C63D9),#2203; Anti-mouse IgG (#7076); Anti-rabbit IgG (#7074) and Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (1:1000, Cell Signaling,#9782) including Vimentin (D21H3) #5741; N-Cadherin (D4R1H)#13116; Claudin-1 (D5H1D)#13255; β-Catenin (D10A8) #8480; ZO-1 (D7D12)#8193; Snail (C15D3)#3879; Slug (C19G7)#9585; ZEB1 (D80D3)#3396; E-Cadherin (24E10) #3195; Anti-rabbit IgG#7074 and GAPDH antibody (1:5000, Cell Signaling, (D16H11)#5174S).

Techniques: Transfection, Luciferase, Activity Assay, Inhibition, Translocation Assay, Western Blot, Immunofluorescence