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: Interaction of PTBP1 and ZMIZ1-AS1 alters the subcellular localization of PTBP1. ( a ) RBPs potentially interacting with ZMIZ1-AS1 were predicted using three databases: RBPsuite ( http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/ ); starBase ( https://rnasysu.com/encori/ ); and catRAPID ( http://service.tartaglialab.com/page/catrapid_omics2_group ). Nineteen RBPs were commonly identified. ( b ) The number of potential PTBP1 binding sites on the ZMIZ1-AS1 transcript was predicted by catRAPID. ( c ) PTBP1 mRNA expression was significantly elevated in osteosarcoma tissues ( n = 103) compared to non-tumor tissues ( n = 15) from the GEO dataset GSE42352 ( p = 0.0182). ( d ) Analysis of PTBP1 expression in normal ( n = 2) and primary sarcoma ( n = 260) tissues from the TCGA database using UALCAN ( https://ualcan.path.uab.edu/ ). ( e ) Kaplan-Meier survival analysis of sarcoma patients based on PTBP1 expression levels (GEPIA2, http://gepia2.cancer-pku.cn/#index ; p = 0.014). ( f ) Western blot analysis of PTBP1 protein expression in osteosarcoma ( n = 5) and paratumor tissues ( n = 5). * p < 0.05; ** p < 0.01 vs. paratumor. ( g ) Western blot analysis of PTBP1 protein expression in normal osteoblasts (hFOB 1.19) and osteosarcoma cells (U2OS, HOS, 143B, MG-63). **** p < 0.0001 vs. hFOB 1.19. ( h ) Prediction of PTBP1 binding sites on the ZMIZ1-AS1 sequence using RBPsuite. ( i - j ) RIP assays demonstrating the enrichment of ZMIZ1-AS1 by PTBP1 in ( i ) 143B and ( j ) HOS cells. *** p < 0.001; **** p < 0.0001 vs. IgG ( k - l ) RNA pulldown assay verifying the enrichment of PTBP1 by the ZMIZ1-AS1 probe in 143B and HOS cells. ( m - n ) Immunofluorescence analysis of PTBP1 subcellular localization in ( m ) 143B and ( n ) HOS cells after ZMIZ1-AS1 knockdown or overexpression. ( o - r ) Western blot analysis of PTBP1 expression in nuclear and cytoplasmic fractions. ( o ) 143B cells with ZMIZ1-AS1 knockdown. ( p ) 143B cells with ZMIZ1-AS1 overexpression. ( q ) HOS cells with ZMIZ1-AS1 knockdown. ( r ) HOS cells with ZMIZ1-AS1 overexpression

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: Binding Assay, Expressing, Western Blot, Sequencing, Immunofluorescence, Knockdown, Over Expression

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