Journal: Frontiers in Oncology

Article Title: MiR-146b overexpression promotes bladder cancer cell growth via the SMAD4/C-MYC/Cyclin D1 axis

doi: 10.3389/fonc.2025.1565638

Figure Lengend Snippet: MiR-146b overexpression inhibited c-myc mRNA transcription in human BCa cells. (A) Relative c-myc mRNA expression detected in UMUC3 (miR-146bi) and UMUC3 (nonsense) cells. (B) UMUC3 (nonsense) versus UMUC3 (miR-146bi) cells were transiently transfected with a c-myc promoter-driven luciferase reporter together with pRL-TK. Transfectants were seeded into 96-well plates to determine c-myc promoter transcriptional activity. pRL-TK was used as the internal control to normalize transfection efficiency. Bars indicate means ± SD from three replicate assays. (C) Potential transcriptional factor binding sites in human c-myc promoter region were analyzed via the TRANSFAC 8.3 engine online. (D, E) Cell lysates from indicated cells were evaluated for Ets1 and SMAD4 protein expression. GAPDH served as the loading control. BCa, bladder cancer. The asterisk (*) indicates a statistically significant difference compared to the control (*p < 0.05).

Article Snippet: The constructs of short hairpin RNA specifically targeting SMAD4 (shSMAD4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Over Expression, Expressing, Transfection, Luciferase, Activity Assay, Control, Binding Assay

Journal: Frontiers in Oncology

Article Title: MiR-146b overexpression promotes bladder cancer cell growth via the SMAD4/C-MYC/Cyclin D1 axis

doi: 10.3389/fonc.2025.1565638

Figure Lengend Snippet: SMAD4 was a miR-146b-regulated transcription factor mediating Cyclin D1 upregulation in human BCa cells. (A) SMAD4 knockdown constructs were stably transfected into UMUC3 (miR-146bi) cells. The SMAD4 knockdown efficiency and its downstream C-MYC, Cyclin D1 expression were assessed by Western blotting. GAPDH was used as a protein loading control. (B) Relative c-myc mRNA expression detected in UMUC3 (miR-146bi/nonsense), UMUC3 (miR-146bi/shSMAD4-1) and UMUC3 (miR-146bi/shSMAD4-2) cells. (C) UMUC3 (miR-146bi/nonsense), UMUC3 (miR-146bi/shSMAD4-1), and UMUC3 (miR-146bi/shSMAD4-2) cells were transiently transfected with a c-myc promoter-driven luciferase reporter together with pRL-TK. Transfectants were seeded into 96-well plates to determine c-myc promoter transcriptional activity. pRL-TK was used as the internal control to normalize transfection efficiency. Bars indicate means ± SD from three replicate assays. (D) The stable transfectants as indicated were subjected to anchorage-independent soft agar growth assay. Representative images of colonies were photographed under an Olympus DP71. (E) The number of colonies was counted with more than 32 cells of each colony, and the results were presented as colonies per 10,000 cells. The bars show mean ± SD from three independent experiments, and the asterisk (*) indicates a significant increase in comparison to nonsense transfectants as indicated (*p < 0.05). BCa, bladder cancer.

Article Snippet: The constructs of short hairpin RNA specifically targeting SMAD4 (shSMAD4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Construct, Stable Transfection, Transfection, Expressing, Western Blot, Control, Luciferase, Activity Assay, Growth Assay, Comparison

Journal: Frontiers in Oncology

Article Title: MiR-146b overexpression promotes bladder cancer cell growth via the SMAD4/C-MYC/Cyclin D1 axis

doi: 10.3389/fonc.2025.1565638

Figure Lengend Snippet: MiR-146b was responsible for destabilization of smad4 mRNA by direct binding to its 3′-UTR. (A) UMUC3 (miR-146bi) and UMUC3 (nonsense) cells were cultured in 6-well plates till cell density reached 70%–80%. Following synchronization for 12 h, the medium was then replaced with 10% FBS DMEM for another 12 h The cells were extracted for total RNA with TRIzol reagent. Real-time PCR was used to determine smad4 mRNA expression, and gapdh was used as an internal control. Data represent mean ± SD (*p < 0.05). (B) UMUC3 (miR-146bi) and UMUC3 (nonsense) cells were seeded into 6-well plates. After synchronization, the indicated cells were treated with Actinomycin D (Act D) for the indicated time points. Total RNA was then isolated and subjected to real-time PCR analysis for mRNA levels of smad4 , and gapdh was used as an internal control. Bars represent mean ± SD from three independent experiments. Student’s t -test was utilized to determine the p-value. The asterisk (*) indicates a significant increase in comparison to UMUC3 (nonsense) cells (*p < 0.05). (C) The pMIR- SMAD4 3′-UTR reporter was transiently transfected into the indicated cells, and luciferase activity of each transfectant was evaluated. The luciferase activity was presented as a relative to nonsense transfectant normalized using pRL-TK as an internal control. The bars show mean ± SD from three independent experiments. The symbol (*) indicates a significant increase in UMUC3 (miR-146bi) in comparison to nonsense transfectant (p < 0.05). (D) The predicted miR-146b binding site existed in the 3′-UTR of smad4 mRNA, and its mutants (MUTs) were generated in the binding site. (E) The pMIR- smad4 3′-UTR reporters (WT and MUT) were co-transfected with pRL-TK into the indicated cells. Twenty-four hours post-transfection, the transfectants were extracted for determination of the luciferase activity, and TK was used as the internal control. Luciferase activity of each transfectant was evaluated, and the results were presented as relative SMAD4 3′-UTR activity. The bars show mean ± SD from three independent experiments. The double asterisk (**) indicates a significant inhibition of 3′-UTR activity in mutant transfectant in comparison to mutant of WT smad4 3′-UTR luciferase reporter transfectant (p < 0.05). (F) The proposed mechanisms underlying miR-146b overexpression in the promotion of human BCa cells growth: miR-146b overexpression destabilizes smad4 mRNA, which further elevates c-myc mRNA transcription, in turn promoting the transcription of cyclin d1 and protein expression, and finally increases the transition of cell cycle G0/G1 phase and the anchorage-independent growth of human BCa cells. FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; BCa, bladder cancer.

Article Snippet: The constructs of short hairpin RNA specifically targeting SMAD4 (shSMAD4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Control, Isolation, Comparison, Transfection, Luciferase, Activity Assay, Generated, Inhibition, Mutagenesis, Over Expression, Modification

Journal: International Journal of Molecular Sciences

Article Title: TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

doi: 10.3390/ijms252111420

Figure Lengend Snippet: TGF-β-induced expression of pancreatic adenocarcinoma upregulated factor (PAUF) is mediated by activating the Smad signaling pathway. ( A ) Different PDAC cell lines were treated with vehicle or TGF-β (10 ng/mL) for 1 h. Phosphorylation and protein levels of TGF-β-mediated Smad signaling molecules were examined using immunoblotting. Densitometry was performed using the ImageJ software ( n = 3). Panc-1 cells were pretreated with SB-431542 (10 μM) for 1 h and stimulated with vehicle or TGF-β for ( B ) 1 or ( C , D ) 24 h. ( B ) Phosphorylation and protein levels of Smads were determined using immunoblotting. ( C ) mRNA and protein levels of PAUF were determined using RT-PCR and immunoblotting, respectively. ( D ) Intracellular PAUF expression was confirmed through immunofluorescence staining with an Alexa 488-labeled anti-PAUF antibody and nuclei were stained with DAPI. Scale bar, 50 μm. Relative PAUF-expressing cells were quantitated in four fields per sample using the ImageJ software ( n = 4). Panc-1 cells were transfected with 100 nM scrambled (Ctrl), Smad2/3, or Smad4 siRNAs alone or in combination, followed by treatment with vehicle or TGF-β for ( E ) 1 or ( F , G ) 24 h. ( E ) Phosphorylation and expression of Smad proteins were measured using immunoblotting. ( F ) mRNA and protein levels of PAUF were determined using RT-PCR and immunoblotting, respectively, and ( G ) secreted PAUF levels were measured in culture supernatants using ELISA ( n = 4). BxPC-3 cells were transfected with control vector pCMV6 (Mock) or Smad4 expression vector pCMV-Smad4 (pcSmad4), followed by treatment with vehicle or TGF-β (10 ng/mL) for ( H ) 1 or ( I , J ) 24 h. ( H ) Smad2/3 phosphorylation and Smad4 expression were determined using immunoblotting. ( I ) PAUF expression was assessed using RT-PCR and immunoblotting. ( J ) Intracellular PAUF levels were visualized using immunofluorescence staining with an Alexa 488-labeled anti-PAUF antibody and nuclei were stained with DAPI. Scale bar, 50 μm. Relative PAUF-expressing cells were quantitated in four fields per sample in a randomized manner using the ImageJ software ( n = 4). Statistical significance was calculated using two-way ANOVA followed by post-hoc multiple comparisons test with Bonferroni correction. Data are presented as the mean ± standard deviation (SD). ns, not statistically significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human TGF-β and TGF-β receptor I kinase inhibitor, SB-431542, were purchased from R&D systems and Sigma-Aldrich (St. Louis, MO, USA), respectively. siRNAs targeting human Smad2/3 (cat# sc-37238), Smad4 (cat# sc-29484), and ZG16B/PAUF (cat# sc-93479) were purchased from Santa Cruz Biotechnology, and pCMV6-XL5 (cat# PCMV6XL5), pCMV-Smad4 (cat# SC116771), pLenti-control (cat# PS100092), and pLenti-PAUF (cat# RC202244L3) vectors were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling, Transfection, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

doi: 10.3390/ijms252111420

Figure Lengend Snippet: Smad-binding element (SBE) is essential for TGF-β-inducibility of the pancreatic adenocarcinoma upregulated factor (PAUF) promoter. ( A ) A schematic representation of the PAUF promoter (−1.7 Kb), highlighting the putative SBE used to analyze the PAUF promoter activity. ( B ) Panc-1 cells were pretreated with SB-431542 (10 μM) for 1 h and subsequently with vehicle or TGF-β (10 ng/mL) for 1 h. Binding of pSamd3 to SBE within the PAUF promoter was determined using the chromatin immunoprecipitation (ChIP) assay. ( C ) Panc-1 cells transfected with the PAUF promoter-Luc vector (pLuc-PAUF) were stimulated with vehicle or TGF-β for 24 h after pretreatment with SB-431542 (10 μM) for 1 h. The promoter activity was determined in cell lysates with a luminometer ( n = 3). ( D ) Panc-1 cells were transfected with scrambled-, Smad2/3-, or Smad4-targeting siRNAs alone or in combination, followed by stimulation with or without TGF-β for 1 h to assess the binding of pSmad3 to SBE within the PAUF promoter using the ChIP analysis. ( E ) Panc-1 cells were transfected with the indicated siRNA alone or in combination with the pLuc-PAUF vector, and then treated with vehicle or TGF-β for 24 h to evaluate the PAUF promoter activity in cell lysates with a luminometer ( n = 3). ( F ) BxPC-3 cells transfected with pCMV6 (Mock) or pCMV-Smad4 (pcSmad4) were stimulated with TGF-β for 24 h. Binding activity of pSmad3 to SBE within the PAUF promoter was analyzed using the ChIP assay. The fold intensity of ChIP enrichment for the SBE motif at −804/−801 was quantified and normalized against the input ( n = 3). ( G ) BxPC-3 cells were transfected with mock or pcSmad4 in combination with the PAUF promoter-Luc vector, and then treated with vehicle or TGF-β for 24 h. The PAUF promoter activity was measured in cell lysates ( n = 3). ( H ) Comparison of the putative SBE motif within the PAUF promoter (−1.7 Kb) with the SBE mutants. ( I ) Wild-type (SBE WT ) and SBE site mutants (SBE Mut and SBE ∆ mut ) of PAUF promoter-Luc vectors were transiently transfected into Panc-1 cells. After 24 h of transfection, the cells were stimulated with vehicle or TGF-β for 24 h and promoter activities were measured in cell lysates ( n = 3). ( J ) BxPC-3 cells were transfected with mock or pcSmad4 in combination with PAUF promoter-Luc vectors (SBE WT , SBE Mut , or SBE ∆ mut ), and then treated with vehicle or TGF-β for 24 h. The promoter activity was determined in cell lysates ( n = 3). Statistical significance was determined using two-way ANOVA followed by post-hoc multiple comparisons test with Bonferroni correction. Data are displayed as the mean ± standard deviation (SD). ns, not statistically significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human TGF-β and TGF-β receptor I kinase inhibitor, SB-431542, were purchased from R&D systems and Sigma-Aldrich (St. Louis, MO, USA), respectively. siRNAs targeting human Smad2/3 (cat# sc-37238), Smad4 (cat# sc-29484), and ZG16B/PAUF (cat# sc-93479) were purchased from Santa Cruz Biotechnology, and pCMV6-XL5 (cat# PCMV6XL5), pCMV-Smad4 (cat# SC116771), pLenti-control (cat# PS100092), and pLenti-PAUF (cat# RC202244L3) vectors were obtained from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Comparison, Standard Deviation