Fig 2: PAICS is involved in tumor growth and epithelial to mesenchymal transition. (A) qPCR analysis of PAICS, SNAI1, SLUG, and Vimentin mRNA in PAICS knockdown or NT-siRNA–treated RT112 cells. (B) Immunoblot analysis showing the levels of PAICS and E-cadherin proteins after PAICS knockdown. (C) Immunostaining of the BLCA cell line RT112 transfected with PAICS and nontargeting siRNA. Red staining represents E-cadherin, and blue represents nuclear staining with DAPI. (D) Tumor growth of VMCUB1 and 5637 PAICS stable knockdown cells or nontargeting control shRNA cells in the CAM tumor assay. Extraembryonic tumors were harvested and weights were measured 72 hours postimplantation of cells. (E) Proposed model of miR-128, PAICS, and EMT regulatory axis in BLCA progression. miR-128 downregulates PAICS expression, and further, PAICS regulates EMT markers SNAI1, VIM, ECAD, and SLUG. PAICS induces cell proliferation, invasion, and tumor growth via these EMT markers.

Fig 3: ADSL is the critical gene targeted by miR-21 in regulating purine metabolism.A Computational prediction of RNA duplex formation between miR-21-3p and ADSL mRNA’s 3’UTR (untranslated region). Mutations generated within the 3’UTR for the luciferase assay are shown in red. WT, wild-type. B Dual-luciferase reporter assay in H1975 cells treated with the miR-21-3p inhibitor. The Firefly luciferase and Renilla luciferase activities were measured 48 h post-co-transfection with the locked nucleic acid (LNA) miR-21-3p inhibitor (anti-miR-21-3p) or a control plasmid (anti-miR-control) and WT or mutant ADSL 3’UTR. N = 3 replicates. C Dual-luciferase reporter assay in H1975 cells treated with the miR-21-5p inhibitor. The Firefly luciferase and Renilla luciferase activities were measured 48 h post-co-transfection with the locked nucleic acid (LNA) miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control) and wild-type (WT) or mutant ADSL 3’UTR. N = 5 replicates. D Western blot analysis and quantification of ADSL in H1975 parental and osimertinib-tolerant (OTR) cells. ß-Actin was used as a loading control. N = 3 replicates. E Western blot analysis and quantification of ADSL in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control). ß-Actin was used as a loading control. N = 2 replicates. F Western blot analysis and quantification of ADSL in parental H1975 cells treated with the LNA inhibitor against miR-21-3p (anti-miR-21-3p), miR-21-5p (anti-miR-21-5p), and a miRNA control (anti-miR-control). ß-Actin was used as a loading control. N = 2 replicates. G A correlation between MIR21 and ADSL gene expression in lung tumor tissues from patients. RNA expression data were extracted from three independent RNA transcriptomics datasets (GSE3141, GSE16534, and GSE8894) by the Lung Cancer Explorer web portal. Each dot represents one patient’s sample. H A diagram describing the purine de novo synthesis pathway and nucleotide cycle reactions. The enzymes in green and blue colors are predicted targets of miR-21-5p and miR-21-3p by the TargetScan analysis. The enzymes in gray color are non-predicted targets of either miR-21-5p or miR-21-3p. I Western blot analysis and quantification of PPAT, GART, PFAS, PAICS, ATIC, AMPD2, and ADSS in H1975 parental and OTR cells. ß-Actin shown in (D) was used as the loading control. N = 2 replicates. J Western blot analysis and quantification of PPAT, GART, PFAS, PAICS, ATIC, AMPD2, and ADSS in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control). ß-Actin shown in (E) was used as the loading control. N = 2 replicates. K Western blot analysis and quantification of MTHFD1 and MTHFD2 in H1975 parental and OTR cells. ß-Actin shown in (D) was used as the loading control. N = 2 replicates. L Western blot analysis and quantification of MTHFD1 and MTHFD2 in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) and a miRNA control plasmid (anti-miR-control). ß-Actin shown in (E) was used as the loading control. N = 2 replicates. M Western blot analysis and quantification of PPAT, GART, and PAICS in H1975 parental cells treated with the LNA inhibitor against miR-21-3p (anti-miR-21-3p), miR-21-5p (anti-miR-21-5p), and a miRNA control (anti-miR-control). ß-Actin shown in (F) was used as the loading control. N = 3 replicates. Data are mean ± s.e.m. and were analyzed with unpaired two-tailed t-test (B–E, I–L); Pearson correlation analysis (G); RM one-way ANOVA (F, M).

Fig 4: PAICS regulates BLCA cell proliferation, colony formation and invasion. (A) Stable knockdown of PAICS reduces cell proliferation of BLCA cell lines VM-CUB1 and 5637. Immunoblot analysis showing PAICS protein in VM-CUB1 and 5637 cells stably transduced with a shRNA specific to PAICS or control nontargeting shRNA. (B) Photographic images of colony formation assay using above test cells. (C) PAICS shRNA-treated BLCA cells showed reduced invasion in Boyden chamber Matrigel invasion assay. Invaded cells were stained with crystal violet.

Fig 5: miR-128 targets and downregulates PAICS in BLCA. (A, B) The predicted miR-128 binding sites in the 3'-UTRs of PAICS. Schematic representation of wild-type and mutant PAICS 3'-UTRs is given, respectively. Red nucleotides represent mutations that are predicted to disrupt potential miR-128 binding. BLCA cells VM-CUB1 and RT112 were treated with pre-miR-128, and (C) immunoblot analysis was performed showing PAICS protein expression, (D) cell proliferation, (E) colony formation, (F) Matrigel invasion, and (G) 3D spheroid formation. (H) Luciferase reporter assay of PAICS-3'-UTR. 293T cells were transfected either with pre-miR-128 or nontargeting pre-miR (NT-pre-miR) along with either PAICS-3'-UTR wild-type or mutant luciferase constructs. pRL-TK vector was used as an internal control.