Fig 2: The effect of YBX1 on the expression of different B7‐H3 isoforms. (A) WB assays show the expression of 4Ig and 2Ig affected by knockdown of YBX1 in A172, LN229 and U251MG cells. ANXA2 and NSUN2 are not affected by YBX1 knockdown. (B) RT‐PCR show the PCR products of B7‐H3 isoform affected by knockdown of YBX1 in A172, LN229 and U251MG cells. P1 primers (top), P2 primers (middle) and β‐actin primers (bottom). (C) WB assays show the expression of 4Ig and 2Ig affected by over‐expression of YBX1 in U87MG cell. ANXA2 and NSUN2 are not affected by over‐expression of YBX1. (D) RT‐PCR show the PCR products of B7‐H3 isoform affected by over‐expression of YBX1 in U87MG cell. P1 primers (top), P2 primers (middle) and β‐actin primers (bottom). (E) WB assays show the substantial interaction with ANXA2 and YBX1 by Flag (ectopic NSUN2) IP in A172 cell. Mutant NSUN2 captures much less ANXA2 and YBX1 compared to wild‐type one. (F) RNA‐EMSA shows the interaction among recombinant human YBX1, ANXA2 and the synthetic partial B7‐H3 RNA with or without 5mC in vitro. ANXA2 appears to have slower mobility due to attaching with RNA in Lane 4, 5 and 9. YBX1 appears to have slower mobility due to attaching with methylated RNA in Lane 6. YBX1 and ANXA2 appear to form one complex and show slowest mobility. All these changes are highlighted by red arrows. (G) Schematic representation shows the regulatory relationship between ANXA2/NSUN2/YBX1 on generation of B7‐H3 isoforms in glioma. In accordance with copyright requirements, we hereby declare that this image is drawn using “FIGDRAW” software. m+, methylated RNA; m−: unmethylated RNA.

Fig 3: HAR1A diminished the malignant behaviors of A549 and H1299 cells by downregulating ANXA2.Stable A549 and H1299 LV-HAR1A cells were further transfected with ANXA2 plasmids. A Representative image of crystal violet-stained A549 and H1299 cells with modified expression of HAR1A and ANXA2 in the transwell assays. Quantification of migrated and invaded cells indicated that transfection of ANXA2 plasmids partially counteracted HAR1A-induced repression of tumor cell migration and invasion. B CCK8 was used to measure proliferation of A549 and H1299 cells transduced with LV-vector, LV- HAR1A+ANXA2 plasmid, and LV- HAR1A+empty plasmid. C Gene set enrichment analysis (GSEA) showed the downstream pathway of ANXA2. D Western blot to determine the protein levels of p65, p-p65, and ANXA2 in A549 and H1299 cells with indicated gene manipulations. Tumor samples were collected from eight NSCLC patients, along with matched normal tissues. E Chest computed tomography imaging of the eight NSCLC patients. The red circles highlight primary lung tumors and the blue arrows indicate regional lymph nodes with pathologically proven metastasis. According to the 8th edition of AJCC TNM staging, N category was defined as N0 (no metastatic lymph node), N1 (metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes), N2 (metastasis in ipsilateral mediastinal and/or subcarinal lymph nodes). F RT-qPCR was used to measure HAR1A in normal tissues versus tumors. G Comparsion of HAR1A in N0 versus N1-2 tumors. H Western blot to compare the ANXA2 levels between N0 and N1-2 tumors. I ANXA2 immunohistochemistry staining in NSCLC tumor samples. J, K Kaplan–Meier survival curves for lung cancer patients dichotomized by the levels of HAR1A or ANXA2, using the K–M plotter. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4: Analysis of HMOX1 and ANXA2 as risk predictors of clinical outcome in PCa. (A) Kaplan–Meier (KM) curves for relapse free survival (RFS) (months) and risk table for PCa patients with high (red) or low (blue) expression of HMOX1 and ANXA2 in Ross-Adams dataset (n = 206). Low expression group was taken as a reference group. Logrank P, HR: Hazard ratio [95% confidence interval] and Cox P are specified. (B) Univariable and multivariable analyses are presented by forest plots for relapse free survival regarding HMOX1 (upper panel) or ANXA2 (lower panel) expression. Multivariable analysis w/Gleason group = adjusted for Gleason groups (6; 7 (3+4); 7 (4+3); 8-10). Multivariable analysis w/PSA = adjusted for PSA serum levels at diagnosis (PSA groups (ng/mL): < 4; 4-10; > 10). Multivariable analysis w/clinical stage = adjusted for patients’ clinical stage. Multivariable analysis w/pathological stage = adjusted for patients’ pathological stage. w/= with. (C) Multivariable analyses based on the mRNA expression of HMOX, ANXA2, Gleason Score, PSA at time of diagnosis, clinical and pathological stage for patients with PCa in the Ross-Adams dataset. (D) Heatmap depicting low (blue) or high (red) HMOX1 and ANXA2 mRNA expression for patients with PCa according to the Ross-Adams dataset. Patient subgroups are presented in black boxes. (E) RFS of patients with low HMOX1 and ANXA2 expression (n = 26) (1), high ANXA2 and low HMOX1 expression (n = 97) (2), low ANXA2 and high HMOX1 expression (n = 3) (3), and high ANXA2 and HMOX1 expression (n = 78) (4) (left panel); RFS of patients with low HMOX1 and ANXA2 expression (n = 26) (1), either high ANXA2 or HMOX1 expression (n = 100) (2), and high ANXA2 and HMOX1 expression (n = 78) (3) (right panel). (F) KM curves for RFS and risk table for PCa patients with high (red) or low (blue) expression of HMOX1, segregated based on low ANXA2 (left panel); and high (red) or low (blue) expression of ANXA2, segregated based on low HMOX1 (right panel). Stata software (StataCorp LLC, Texas, USA) was used to assess the patient’s overall and RFS and to generate Kaplan–Meier curves. Minimal p-value approach from the Cutoff Finder tool [31] was used to stratify patients in two groups based on the gene expression levels. For univariable and multivariable analyses of prognostic factors, Log-rank test and Cox proportional hazard model regression was employed. Statistical significance was set at p < 0.05.

Fig 5: Schematic representation of ANXA2 and HO-1 expression in PCa and metastasis. ANXA2 expression is downregulated in prostate carcinoma compared with the normal gland. In PCa bone metastasis there is an increase in ANXA2 expression compared with the primary tumor site. Similarly, the presence of bone cells in the co-culture induces an increase in ANXA2 expression in the tumor cell, reflecting that the bone niche modulates the expression of ANXA2 in the prostate tumor cell. In turn, the presence of tumor cells triggers a change in the sub-cellular localization of ANXA2 in the pre-osteoclastic cells. This effect is hampered under HO-1 induction in PC3 cells. This phenomenon could be explained, at least in part, by the differences in the availability of free calcium in both conditions and the presence of urokinase (uPA) in the conditioned media.