Fig. 4: YBX1 recognizes NSUN2-deposited m⁵C to promote SERPINB5 mRNA stability and expression in cervical cancer.

A Box plots showing YBX1 expression levels measured by RNA-seq in control (n = 7) and cervical cancer (n = 9) samples. B Representative immunohistochemical staining of YBX1 in control and cervical cancer tissues (left). Scale bars, 200 μm. Quantification by H-score shows significantly higher YBX1 protein levels in cancer tissues (n = 8 per group) (right). Data are presented as box plots showing the median and min to max range. C Representative spatial mapping of YBX1 expression in control, tumor periphery, and tumor core. The pie chart quantifies the percentage of YBX1⁺ cells. D Correlation between YBX1 and SERPINB5 mRNA expression in cervical cancer samples. E qPCR analysis of YBX1 mRNA levels after siRNA-mediated knockdown (siYBX1 #1 and siYBX1 #2) in HeLa cells (n = 3). Data are presented as mean ± SD. F Relative expression of SERPINB5 mRNAs following YBX1 knockdown in HeLa cells (n = 3). Data are presented as mean ± SD. G RNA stability assay of SERPINB5 mRNA in HeLa cells treated with actinomycin D following negative control (siNC) or YBX1 siRNA knockdown. Remaining mRNA levels were quantified by qPCR at the indicated time points (n = 3). Data are presented as mean ± SD. H YBX1-RIP-qPCR analysis was performed using an anti-YBX1 antibody to immunoprecipitate HA-tagged YBX1, and the enrichment of SERPINB5 mRNA was subsequently assessed (left) (n = 3). Data are presented as mean ± SD. Immunoblot analysis confirmed the presence of YBX1 protein in both input and immunoprecipitated (IP) samples, with IgG used as a negative control (right). I Schematic illustration of the three predominant m⁵C methylation sites identified within SERPINB5 transcripts, including one site located in the CDS and two sites within the 3’UTR, along with their respective methylation frequencies. J Schematic of the SERPINB5 CDS luciferase reporter containing a single m⁵C site mutation (C1018T). K HeLa cells were transfected with pGL.3.0-CMV-Luc or pGL3.0-CMV-SERPINB5-CDS-Luc (WT or C1018T mutant), together with Renilla luciferase (RL-TK), followed by treatment with YBX1 siRNA or control siRNA treatment. Luciferase activity was measured 48 h later, and firefly luciferase activity was normalized to Renilla luciferase activity (n = 3). Data are presented as mean ± SD. L Schematic of the SERPINB5 3’UTR luciferase reporter containing double m⁵C site mutations (C1506T/C2245T). M HeLa cells were transfected with pGL.3.0-CMV-Luc or pGL3.0-CMV-SERPINB5-3’UTR-Luc (WT or double mutant), together with RL-TK, and treated with YBX1 siRNA or control siRNA. Firefly luciferase activity was measured and normalized to Renilla luciferase activity 48 h post-transfection (n = 3). Data are presented as mean ± SD. N Immunofluorescence microscopy of cervical cancer tissues stained with DAPI (blue), YBX1 (purple), SERPINB5 (green), and NSUN2 (red) antibodies. Left: merged image showing co-localization (scale bar, 500 μm). Right: individual and pairwise merged images showing distribution patterns of NSUN2, YBX1, and SERPINB5 (scale bar, 100 μm). Statistical analyses were performed using two-tailed unpaired t test for (A, B-right, E, F, H-left, K, M), Pearson correlation for association analyses (D), and two-way ANOVA followed by multiple comparisons test for (G). NS not significant for P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.