Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALKBH5 demethylation modification of SE-lncRNA ZMIZ1-AS1 promotes FGFR1-mediated proliferation and invasive metastasis in osteosarcoma

doi: 10.1007/s00018-025-05969-2

Figure Lengend Snippet: ZMIZ1-AS1 promotes FGFR1 stability in a PTBP1-dependent manner. ( a ) Correlation between ZMIZ1-AS1 and FGFR1 expression in osteosarcoma tissues ( n = 87) from the TARGET-OS database ( R = 0.4789, p < 0.0001). ( b ) Correlation between PTBP1 and FGFR1 expression in sarcoma tissues ( n = 260) from the TCGA database analyzed using GEPIA2 ( http://gepia2.cancer-pku.cn/#correlation ) ( R = 0.36, p = 1.3 × 10⁻⁹). ( c ) Correlation between FGFR1 and PTBP1 expression in osteosarcoma cells from the GEO dataset GSE33458 ( n = 17) ( R = 0.7708, p = 0.0002). ( d ) Number of predicted PTBP1 binding sites on the FGFR1 transcript, as determined by catRAPID. ( e ) Analysis of FGFR1 expression in normal ( n = 2) and primary sarcoma ( n = 260) tissues from the TCGA database using UALCAN ( https://ualcan.path.uab.edu/ ). ( f ) Kaplan-Meier survival analysis of sarcoma patients based on FGFR1 expression levels (GEPIA2, http://gepia2.cancer-pku.cn/#index ; p = 0.023). ( g ) FGFR1 mRNA expression levels in osteosarcoma ( n = 50) and paired paratumor tissues ( n = 50), measured by RT–qPCR. **** p < 0.0001. ( h ) FGFR1 protein expression levels in osteosarcoma ( n = 5) and paired paratumor tissues ( n = 5), analyzed by Western blotting. *** p < 0.001; **** p < 0.0001. (i-j) FGFR1 ( i ) mRNA and ( j ) protein expression in normal osteoblasts (hFOB 1.19) and osteosarcoma cells (U2OS, HOS, 143B, MG-63) was detected by RT-qPCR and Western blotting, respectively. ** p < 0.01; **** p < 0.0001 vs. hFOB 1.19. (k-l) RNA immunoprecipitation (RIP) assays showing enrichment of FGFR1 mRNA by anti-PTBP1 antibody in ( k ) 143B and ( l ) HOS cells. * p < 0.05, *** p < 0.001 vs. IgG. ( m , n ) RNA pulldown assay using a biotinylated FGFR1 probe confirms the direct binding between PTBP1 and FGFR1 mRNA in ( m ) 143B and ( n ) HOS cells. ( o , p ) FGFR1 mRNA expression following transfection with the indicated constructs in ( o ) 143B and ( p ) HOS cells. ** p < 0.01, **** p < 0.0001 vs. pcDNA; ### p < 0.001, #### p < 0.0001 vs. oeZMIZ1-AS1 + siNC. ( q – r ) FGFR1 protein expression under the same conditions as in ( o – p ). **** p < 0.0001 vs. pcDNA; ### p < 0.001, #### p < 0.0001 vs. oeZMIZ1-AS1 + siNC. ( s - t ) mRNA stability assay measuring the half-life of FGFR1 mRNA after treatment with actinomycin D (2 µg/mL) in ( s ) 143B and ( t ) HOS cells transfected as indicated. **** p < 0.0001 vs. pcDNA; #### p < 0.0001 vs. oeZMIZ1-AS1 + siNC. ( u ) Colony formation assay assessing the proliferation of 143B and HOS cells under the indicated conditions. *** p < 0.001 vs. pcDNA; #### p < 0.0001 vs. oeZMIZ1-AS1 + siNC. ( v , w ) Transwell assays evaluating the migratory and invasive capacities of ( v ) 143B and ( w ) HOS cells under the indicated conditions. **** p < 0.0001 vs. pcDNA; #### p < 0.0001 vs. oeZMIZ1-AS1 + siNC

Article Snippet: The membranes were blocked with NcmBlot blocking buffer (# P30500 ) for 15 min and subsequently incubated overnight at 4 °C with the following primary antibodies: FOSL1 (1:1000; Abcam, #ab252421), ALKBH5 (1:5000; Proteintech, #67811-1-Ig), PTBP1 (1:4000; Proteintech, #12582-1-AP), and FGFR1 (1:2000; Proteintech, #60325-1-Ig).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, RNA Immunoprecipitation, Transfection, Construct, Stability Assay, Colony Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALKBH5 demethylation modification of SE-lncRNA ZMIZ1-AS1 promotes FGFR1-mediated proliferation and invasive metastasis in osteosarcoma

doi: 10.1007/s00018-025-05969-2

Figure Lengend Snippet: ZMIZ1-AS1 promotes tumor growth and lung metastasis in vivo by modulating the PTBP1/FGFR1 axis. ( a ) Representative photographs of subcutaneous tumors resected from nude mice injected with control or oeZMIZ1-AS1 143B cells. ( b - c ) ( b ) Final tumor volumes and ( c ) tumor growth curves of xenografts in the indicated groups. **** p < 0.0001 vs. vector. ( d ) Representative computed tomography (CT) scans showing subcutaneous tumors in mice from the indicated groups. ( e - f ) Relative mRNA expression levels of ( e ) ZMIZ1-AS1 and ( f ) FGFR1 in subcutaneous tumor tissues, measured by RT–qPCR. **** p < 0.0001 vs. vector group. ( g ) Representative IHC staining images and quantification of FGFR1 protein in subcutaneous tumor tissues. ( h ) Western blot analysis of FGFR1 protein expression in subcutaneous tumor tissues. *** p < 0.001, **** p < 0.0001 vs. vector group. ( i ) Subcellular distribution of PTBP1 in subcutaneous tumor tissues assessed by Western blot analysis of nuclear and cytoplasmic fractions. Lamin B1 and GAPDH serve as markers for the nucleus and cytoplasm, respectively. ( j ) Representative photographs of lungs (left) and H&E staining of lung sections (right) from mice in the indicated groups. ( k ) Representative computed tomography (CT) scans of mouse chest cavities. ( l ) Quantification of lung metastatic nodules in the indicated groups. ** p < 0.01 vs. vector group. ( m - n ) Relative mRNA expression levels of ( m ) ZMIZ1-AS1 and ( n ) FGFR1 in lung metastatic nodules, measured by RT–qPCR. **** p < 0.0001 vs. vector group. ( o ) Western blot analysis of FGFR1 protein expression in lung metastatic nodules. ** p < 0.01 vs. vector. ( p ) Subcellular distribution of PTBP1 in lung metastatic nodules assessed by Western blot analysis of nuclear and cytoplasmic fractions. Lamin B1 and GAPDH serve as markers for the nucleus and cytoplasm, respectively

Article Snippet: The membranes were blocked with NcmBlot blocking buffer (# P30500 ) for 15 min and subsequently incubated overnight at 4 °C with the following primary antibodies: FOSL1 (1:1000; Abcam, #ab252421), ALKBH5 (1:5000; Proteintech, #67811-1-Ig), PTBP1 (1:4000; Proteintech, #12582-1-AP), and FGFR1 (1:2000; Proteintech, #60325-1-Ig).

Techniques: In Vivo, Injection, Control, Plasmid Preparation, Computed Tomography, Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: ALKBH5 demethylation modification of SE-lncRNA ZMIZ1-AS1 promotes FGFR1-mediated proliferation and invasive metastasis in osteosarcoma

doi: 10.1007/s00018-025-05969-2

Figure Lengend Snippet: Combined inhibition of ALKBH5 and FGFR1 synergistically suppresses ZMIZ1-AS1-driven tumor growth and metastasis. (a-b) Dose-response curves and half-maximal inhibitory concentrations (IC₅₀) of ( a ) the ALKBH5 inhibitor 18 L and ( b ) the FGFR1–3 inhibitor BGJ398 (infigratinib) in ZMIZ1-AS1-overexpressing 143B cells. ( c ) Cell viability assessed by CCK-8 assay after treatment with 18 L, BGJ398, or their combination (2.5:1 ratio) for 72 h. ( d ) Combination index (CI) plot calculated by the Chou-Talalay method using CalcuSyn software. CI < 1 indicates synergy. ( e , f ) ( e ) Final tumor volumes and ( f ) tumor growth curves in nude mice bearing oeZMIZ1-AS1 xenografts treated with vehicle, 18 L (20 mg/kg), BGJ398 (10 mg/kg), or the combination. *** p < 0.001, **** p < 0.0001 vs. vehicle; ## p < 0.01 vs. single-agent groups. ( g ) Relative mRNA expression levels of ZMIZ1-AS1 in subcutaneous tumor tissues from the indicated treatment groups, measured by RT–qPCR. ns, not significant; **** p < 0.0001 vs. vehicle. ( h ) MeRIP-qPCR analysis of m⁶A modification levels on ZMIZ1-AS1 in subcutaneous tumor tissues from mice treated with vehicle or 18 L. **** p < 0.0001 vs. vehicle. ( i ) Relative mRNA expression levels of FGFR1 in subcutaneous tumor tissues from the indicated treatment groups, measured by RT–qPCR. ns, not significant; ** p < 0.01, *** p < 0.001 vs. vehicle. ( j ) Western blot analysis of FGFR1 protein expression in subcutaneous tumor tissues from the indicated treatment groups. **** p < 0.0001 vs. vehicle. ( k ) Representative photographs of lungs (top) and H&E-stained sections of lung metastatic nodules (bottom) from mice in the indicated treatment groups. ( l ) Quantification of lung metastatic nodules in the indicated treatment groups. **** p < 0.0001 vs. vehicle; # p < 0.05 vs. single-agent groups. ( m ) RT-qPCR analysis of ZMIZ1-AS1 mRNA expression levels in lung metastatic nodules. ns, not significant; **** p < 0.0001 vs. vehicle. ( n ) MeRIP-qPCR analysis of m⁶A modification levels on ZMIZ1-AS1 in lung metastatic nodules from mice treated with vehicle or 18 L. *** p < 0.001 vs. vehicle. ( o ) RT-qPCR analysis of FGFR1 mRNA expression levels in lung metastatic nodules. ns, not significant; **** p < 0.0001 vs. vehicle. ( p ) Western blot analysis of FGFR1 protein expression in lung metastatic nodules from the indicated treatment groups. **** p < 0.0001 vs. vehicle

Article Snippet: The membranes were blocked with NcmBlot blocking buffer (# P30500 ) for 15 min and subsequently incubated overnight at 4 °C with the following primary antibodies: FOSL1 (1:1000; Abcam, #ab252421), ALKBH5 (1:5000; Proteintech, #67811-1-Ig), PTBP1 (1:4000; Proteintech, #12582-1-AP), and FGFR1 (1:2000; Proteintech, #60325-1-Ig).

Techniques: Inhibition, CCK-8 Assay, Software, Expressing, Quantitative RT-PCR, Modification, Western Blot, Staining