Journal: Non-coding RNA Research

Article Title: SNHG5 enhances colorectal cancer metastasis through RNA–protein interaction with GNB2 and activation of canonical Wnt signaling

doi: 10.1016/j.ncrna.2025.12.002

Figure Lengend Snippet: Highly metastatic MC38-derived CRC cells exhibit enhanced proliferative, migratory, and invasive properties with mesenchymal characteristics . (A) CCK-8 assay demonstrated significantly higher proliferative capacity in the highly metastatic MC38-F3 subline compared to the parental low-metastatic MC38-F0 cells over a 96-h time course. (B) Representative colony formation images and quantification revealed increased clonogenicity in the MC38-F3 subline. (C) Wound healing assays indicated enhanced migratory ability in MC38-F3 cells at 48 h post-scratch. (D) Transwell migration and Matrigel-coated invasion assays showed that MC38-F3 cells exhibited significantly increased motility and invasiveness. Scale bars = 100 μm. (E) Western blot analysis revealed downregulation of the epithelial marker E-cadherin and upregulation of mesenchymal markers N-cadherin, Vimentin, and Slug in MC38-F3 cells. (F) RT-qPCR analysis confirmed significant upregulation of EMT-associated transcription factors (Snail, Slug, Twist1, ZEB1, ZEB2) in MC38-F3 cells. (G) Immunofluorescence staining corroborated the EMT phenotype, showing reduced E-cadherin and increased Vimentin expression in MC38-F3 cells. Nuclei were counterstained with DAPI. Scale bars = 20 μm. Data are presented as mean ± SEM from at least three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 (Student's t -test).

Article Snippet: Membranes were blocked in 5 % non-fat dry milk diluted in TBST (Tris-buffered saline containing 0.1 % Tween-20) for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: GNB2 (1:1000; rabbit monoclonal, clone EP3262Y, Abcam, ab108504), E-cadherin (1:1000; rabbit monoclonal, CST, #14472), N-cadherin (1:1000; rabbit monoclonal, CST, #13116), Vimentin (1:1000; rabbit polyclonal, Proteintech, 10366-1-AP), Slug (1:1000; rabbit monoclonal, CST, #9585) and GAPDH (1:5000; mouse monoclonal, Proteintech, 60004-1-Ig) as a loading control.

Techniques: Derivative Assay, CCK-8 Assay, Migration, Western Blot, Marker, Quantitative RT-PCR, Immunofluorescence, Staining, Expressing

Journal: Non-coding RNA Research

Article Title: SNHG5 enhances colorectal cancer metastasis through RNA–protein interaction with GNB2 and activation of canonical Wnt signaling

doi: 10.1016/j.ncrna.2025.12.002

Figure Lengend Snippet: The Snhg5–GNB2 axis promotes EMT and activates Wnt/β-catenin signaling in CRC cells . (A) Immunohistochemistry of liver metastatic tissues revealed that Snhg5 knockdown reduced β-catenin and Vimentin expression while increasing E-cadherin levels. These changes were partially reversed by GNB2 overexpression. Quantification of IHC signal intensity (IOD) supported the observed protein alterations. Scale bars = 100 μm. (B, D) Western blot analysis of MC38-F0 (B) and MC38-F3 (D) cells showed that Snhg5 silencing increased E-cadherin expression while decreasing N-cadherin, Vimentin, and Slug levels. GNB2 overexpression reversed these EMT-associated changes. (C, E) RT-qPCR confirmed that knockdown of Snhg5 suppressed the transcription of key EMT regulators (Snail, Slug, Twist1, ZEB1, ZEB2), which was restored by GNB2 overexpression in both cell models. (F, H) Western blot analysis of Wnt signaling components revealed that Snhg5 knockdown reduced total β-catenin and phosphorylated GSK-3β (Ser9) levels, with no significant change in total GSK-3β. GNB2 overexpression reversed these effects in both MC38-F0 and F3 cells. (G, I) RT-qPCR analysis demonstrated that Snhg5 depletion led to reduced expression of canonical Wnt target genes (AXIN2, c-MYC, Cyclin D1), which was significantly restored by GNB2. Data are presented as mean ± SEM from at least three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 (one-way ANOVA with Tukey's post hoc test).

Article Snippet: Membranes were blocked in 5 % non-fat dry milk diluted in TBST (Tris-buffered saline containing 0.1 % Tween-20) for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: GNB2 (1:1000; rabbit monoclonal, clone EP3262Y, Abcam, ab108504), E-cadherin (1:1000; rabbit monoclonal, CST, #14472), N-cadherin (1:1000; rabbit monoclonal, CST, #13116), Vimentin (1:1000; rabbit polyclonal, Proteintech, 10366-1-AP), Slug (1:1000; rabbit monoclonal, CST, #9585) and GAPDH (1:5000; mouse monoclonal, Proteintech, 60004-1-Ig) as a loading control.

Techniques: Immunohistochemistry, Knockdown, Expressing, Over Expression, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Determination of the molecular mechanism of WISP1v1 on the cell invasion in the bladder cancer cells. (A) The protein levels of NDRG1, KAI1, and Maspin of HT-DNA and HT-WISP1v1 cells were determined by immunoblot assays. (B) The luciferase activity of the NDRG1 reporter vector was shown after transiently overexpressing various amounts of WISP1v1 expression vectors in HT1376 and T24 cells, as indicated. (C) The luciferase activity of KAI1 and Maspin reporter vectors was shown as indicated after transiently overexpressing various dosages of WISP1v1 expression vectors in HT1376 cells. (D) The luciferase activity of NDRG1, KAI1, and Maspin reporter vectors, respectively, after transiently overexpressed pcDNA, WISP1v1, and WISP1v2 expression vectors as indicated in HT1376 cells. (E) The mRNA levels of the E-cadherin, N-cadherin, Slug, Snail, and Vimentin were determined by RT-qPCR. Data are presented as target genes/β-actin of HT-WISP1v1 cells relative to HT-DNA cells. The mRNA levels of the WISP1 (F), NDRG1, KAI1, Maspin, and IL-6 (G) after transient overexpression of WISP1v1 in T24 cells were determined by RT-qPCR. Data are presented as target genes/β-actin of T24-WISP1v1 cells relative to T24-DNA cells. (H) The invasion ability of T24 cells after the ectopic overexpression of WISP1v1 was determined by in vitro Matrigel invasion assays (± SE; n = 4). The ability of the migration (I) and quantification analysis (I) of T24 cells after being treated with the supernatant from 293T-DNA and 293T-WISP1v1 cells was determined by wound healing assays. The white line indicated the average of the leading cellular edges, and the wound area size was calculated by Image J. * p < 0.05, ** p < 0.01.

Article Snippet: TaqManTM gene expression master mix and polymerase chain reaction (PCR) FAM dye-labeled TaqMan MGB probes for human WISP1 (Hs04234730_m1 for total isoforms and Hs00180245 for WISP1v1), α-SMA (Hs00426835_g1), IL-6 (Hs00985639_m1), GDF15 (Hs00171132_m1), CXCL5 (Hs01099660_g1), SDF-1/CXCL12 (Hs03676656_m1), NDRG1 (Hs00608387_m1), KAI1 (Hs00356310_m1), Maspin (Hs00985283_m1), E-cadherin (Hs01023894_m1), N-cadherin (Hs00169953_m1), Snail (Hs00195591_m1), Slug (Hs00161904_m1), Vimentin (Hs00185584_m1), and β-actin (Hs01060665_g1) were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania).

Techniques: Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, In Vitro, Migration