Fig 2: LMX1B is upregulated in PCa and modulates the tumorigenic properties and immune escape of PCa. (a) Relative expression of LMX1B in thirty pairs of PCa tumor tissues and comparative normal tissues. (b) Protein expression of LMX1B in randomly selected five pairs of tumor tissues and comparative normal tissues. (c) Relative expression of LMX1B in PCa cell lines (RWPE-1, DU145, PC-3, LNCap, and 22Rv1 cell lines) with expression in RWPE-1 cells used as the control. (d and e) Loss-of-function cell models were constructed via transfecting si-NC, si-LMX1B#1, and si-LMX1B#2 into PC-3 and LNCap cells, respectively. The mRNA and protein expression of LMX1B were measured by qRT-PCR and western blot. (f and g) Cell proliferation was detected by CCK-8. (h and i) EdU assay to evaluate cell proliferation. (j and k) Cell migration was assessed by the transwell assay. (l) VEGF content of PCa cell supernatants. (m) IL-10 content of PCa cell supernatants. (n) TGF-β1 content of PCa cell supernatants. (o and p) The cytotoxicity of CIK cells to PC-3 and LNCap cells was determined via CCK-8 assay with results from the si-NC group as control. Data are presented as mean ± standard error. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 3: 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 4: Inhibition of the NMD restores the expression of the mutated allele of LMX1B in the affected members of Family 1 cells.a Lymphoblastoid cell lines derived from B481 proband and his parents (C007 and C008) were treated 24 h with 10 mM of Sodium butyrate and overnight with 200 µg/ml of Puromycin. cDNA was obtained after retro-transcription of 2 µg of RNA and a fragment including the 5’UTR region and the first 4 exons of LMX1B was amplified by PCR. The housekeeping gene GAPDH was used to check the retro-transcription efficiency. b Details of the electropherograms showing the variant −174C>T with (NB/P) or without (NB) the cotreatment with Puromycin. The position of the variant is highlighted by red arrows and the polymorphism rs2277158 A>G by blue stars. un untreated cells, NB Sodium butyrate treated cells, NB/P cells treated with Sodium butyrate and Puromycin, mw molecular weight, – PCR no template control. Image created with BioRender.com.

Fig 5: The identified LMX1B 5’UTR variants impair gene expression at post-transcriptional level.a Workflow for the functional study of LMX1B 5’UTR variants at post transcriptional level as assessed by reporter gene assay (see text for details). b Histograms represent the luciferase activity measured after transient transfection of the different pGL3.PV expression constructs, schematically reported on the left part of the panel. The luciferase values were normalized for transfection efficiency by co-transfection with a Renilla reporter control construct (see Methods for details) and are represented as the fold activation compared to the vector carrying the WT 5’UTR of LMX1B (value 1). Values represent the mean +/−the standard deviation of at least n = 4 biological replicates performed in technical triplicate or quadruplicate (p < 0.001***). EV, luciferase reporter vector without the 5’UTR sequence; WT, wild-type LMX1B 5’UTR. c Workflow for the functional study of LMX1B 5’UTR variants at post transcriptional level as assessed by western blot analysis (see text for details). d Hek-293 cells were transfected with pRP containing the LMX1B cDNA with or without the 5’UTR sequence, wild type or mutated. The expression level of the protein was evaluated by Western Blot, by using anti-Flag and anti-LMX1B primary antibody. Arrows indicate the band corresponding to LMX1B protein (60 kDa) and a non-specific band appearing only in cells transfected with the 5’UTR constructs. Densitometric analysis of LMX1B protein levels was obtained by the anti-Flag and anti-LMX1B normalized on the signal derived from anti-GAPDH antibody. Histograms represent the mean +/− the standard deviation of 6 independent experiments and represent the values compared to the vector without the 5’UTR (value 1). p < 0.01§, p < 0.001# (Fig. 5d right panel). un, untransfected cells; no UTR, vector containing the LMX1B cDNA without 5’UTR sequence; UTRwt, expression vector containing the LMX1B cDNA with the wild type 5’UTR sequence; UTR−174, expression vector carrying the 5’UTR −174C > T variant; UTR−226, expression vector with the 5’UTR −226G>A substitution. Image created with BioRender.com.