Fig 2: HECTD1 is crucial for the functional role of LMX1B in PCa. (a) HECTD1 expression in vector and circGFRA1 transfected cells measured by qRT-PCR and western blotting using the vector group as control. (b) HECTD1 expression in circGFRA1-si-NC, circGFRA1-si-HECTD1#1, and circGFRA1-si-HECTD1#2 transfected cells measured by qRT-PCR and western blotting. (c) Cell proliferation was detected by CCK-8. (d and e) EdU assay was performed to evaluate cell proliferation. (f and g) Cell migration assessed by the transwell assay. (h) VEGF content of PCa cell supernatants. (i) IL-10 content of PCa cell supernatants. (j) TGF-β1 content of PCa cell supernatants. (k and l) The cytotoxicity of CIK cells to PC-3 and LNCap cells was determined via CCK-8 assay using the circGFRA1-si-NC group as control. Data are presented as mean ± standard error. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 3: LMX1B binds to circGFRA1 and upregulates circGFRA1 expression in PCa cells. (a) Relative circGFRA1 expression in PC-3 and LNCap cells upon 0/20/50/100 ng LMX1B transfection and circGFRA1 expression upon 0 ng LMX1B transfection were used as control. (b) Relative circGFRA1 expression in PCa cells upon si-NC, si-LMX1B#1, and si-LMX1B#2 transfection results from si-NC group was used as control. (c) The predicted binding motif of LMX1B obtained from JASPAR. (d) The predicted binding motif of GFRA1 promoter regions obtained from JASPAR. (e and f) ChIP analysis of enrichment of LMX1B on the GFRA1 promoter in PC-3 and LNCap cells. IgG was used as a negative control. (g and h) Quantification of the luciferase activity of the wild-type or mutant GFRA1 promoter reporter in PC-3 and LNCap cells. Data are presented as mean ± standard error. ∗P < 0.05, ∗∗∗P < 0.001.

Fig 4: Analysis of LMX1B cDNA.a Sodium butyrate induces LMX1B gene expression in lymphoblastoid cell lines derived from B481 proband and his parents (C007 and C008). Indeed, an RT-PCR product of 537 bp spanning from the 5’UTR region to exon 2 of LMX1B is well detectable in lymphoblasts treated for 24 h with 10 mM Sodium butyrate and in Hek-293 used as positive control, but not in untreated cells. The housekeeping gene GAPDH was used to check the retro-transcription efficiency. b The variant −174C>T likely impairs the expression of the carrying transcript as assessed by comparing Sanger sequencing of PCR products obtained from genomic versus cDNA. The position of the variant is highlighted by red arrows and that of the A>G polymorphism by blue stars (see text for details). Mw molecular weight, un untreated cells, NB Sodium butyrate, – PCR reaction negative control (no template). Image created with BioRender.com.

Fig 5: Functional evaluation of the newly generated uORF3 and uORF4.a Schematic representation of the strategy we applied to investigate the expression of the new uORFs. We inserted two nucleotides (AG) in position −143 upstream the main LMX1B ATG, to put the new uORFs in frame with the main one in pRP-CMV/5’UTR (hLMX1B) expression vectors, WT and mutated versions. b After transfection of the different plasmids in Hek-293 cells, the expression of the different proteins was evaluated both with anti-LMX1B antibody (left panel) and an anti-FLAG antibody (right panel). The black arrowhead indicates the LMX1B protein and the red and green stars the products of the uORF3 and uORF4 translation when they are placed in frame with the principal ATG. The blot image is representative of three independent experiments. un, untransfected cells; noUTR, vector containing the LMX1B cDNA from ATG; UTRwt, vector containing the WT 5’UTR region and the cDNA of LMX1B; UTR−174, vector containing the −174C>T variant; and the cDNA of LMX1B; UTR−226, vector carrying −226G>A substitution; insAG, expression vectors with the AG insertion in position −143. Image created with BioRender.com.