Fig. 4: circTGFBR2(3-6) binds to and stabilizes TGFBR1 mRNA.

A RT-qPCR analysis of TGFBR1 mRNA expression in MDA-MB-231 cells upon shRNA-mediated circTGFBR2(3-6) knockdown. Data are presented as the mean ± SEM from six biological replicates, with significance assessed using a two-tailed unpaired Student’s t-test. B Effect of shRNA-mediated circTGFBR2(3-6) knockdown on TGFBR1 protein expression in MDA-MB-231 cells. GAPDH, loading control. Data are presented as the mean ± SEM from five independent experiments, with significance assessed using a two-tailed paired Student’s t-test. C Correlation between circTGFBR2(3-6) and TGFBR1 mRNA expression across seven triple-negative breast cancer (TNBC) cell lines. Pearson’s r and two-tailed p value were used to assess correlation. TGFBR1 mRNA stability upon shRNA-mediated circTGFBR2(3-6) knockdown (D) or circTGFBR2(3-6) ectopic expression (E), measured by time-course experiments using actinomycin D (ActD). RT-qPCR data are presented as the mean ± SEM from three biological replicates, with significance assessed using multiple unpaired t-tests. F Schematic representation of a circTGFBR2(3-6) mutant in which all ATG codons mutated to ATT codons (circTGFBR2(3-6) ATG MUT). G Effect of circTGFBR2(3-6) and circTGFBR2(3-6) ATG MUT ectopic expression on TGF-β-induced p-SMAD2 response in MDA-MB-231 cells. Quantitative of p-SMAD2 abundance relative to t-SMAD2 is shown as mean ± SEM from three independent experiments. GAPDH, loading control. Significance was calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. RT-qPCR (H) and Western blotting (I) analysis of TGFBR1 mRNA and protein expression in MDA-MB-231 cells (WT and TGFBR2 Alu Del-1) upon siRNA-mediated AGO2 knockdown. GAPDH, loading control. Data in (H) are expressed as mean ± SEM from three biological replicates, with significance assessed using two-way ANOVA followed by uncorrected Fisher’s LSD test. J Schematic representation of RNA pull-down of circTGFBR2(3-6). K Interaction between circTGFBR2(3-6) and TGFBR1 mRNA in MDA-MB-231 cells, analyzed by RNA pull-down followed by RT-qPCR. Data represent mean ± SEM from three biological replicates, with significance assessed using a two-tailed unpaired Student’s t-test. A LacZ-targeting probe served as a control. L Effect of TGFBR1 CDS ectopic expression on TGF-β-induced p-SMAD2 response in MDA-MB-231 cells upon shRNA-mediated circTGFBR2(3-6) knockdown. An asterisk (*) indicates a non-specific band. GAPDH, loading control. M Effect of TGFBR1 CDS ectopic expression on MDA-MB-231 cell migration upon shRNA-mediated circTGFBR2(3-6) knockdown, measured using a transwell assay. Data are presented as mean ± SEM from five biological replicates. Significance was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test. N Effect of TGFBR1 CDS ectopic expression on TGF-β-induced EMT marker expression in MCF10A-M2 cells upon shRNA-mediated circTGFBR2(3-6) knockdown. GAPDH, loading control. O Proposed working model. circTGFBR2(3-6) interacts with and stabilizes TGFBR1 mRNA, resulting in the enhancement of TGF-β/SMAD signaling, as well as TGF-β-induced EMT and cell migration.