M6A-mediated-upregulation of lncRNA BLACAT3 promotes bladder cancer angiogenesis and hematogenous metastasis through YBX3 nuclear shuttling and enhancing NCF2 transcription

Abstract

Lymphatic metastasis is recognized as the leading manner of metastasis in bladder cancer (BLCa), but hematogenous metastasis accounts for a majority of cancer-associated deaths. The past two decades have witnessed tremendous attention in long non-coding RNAs (lncRNAs), which are a new hope for the development of targeted drug therapy for metastatic cancers; however, the underlying mechanism of lncRNAs involved in BLCa hematogenous metastasis remains to be elucidated. Here, we identified BLCa-associated transcript 3 (BLACAT3), a lncRNA, which was aberrantly upregulated in BLCa and corelated with poor prognosis of patients with muscle-invasive bladder cancer. Methodologically, m6A epitranscriptomic microarray, RNA sequencing and mass spectrometry (MS) were used to screen the key molecules of the regulatory axis. Functional assays, animal models and clinical samples were used to explore the roles of BLACAT3 in BLCa in vitro and in vivo. Mechanistically, m6A modification contributes to BLACAT3 upregulation by stabilizing RNA structure. BLACAT3 recruits YBX3 to shuttle into the nucleus, synergistically enhances NCF2 transcription, and promotes BLCa angiogenesis and hematogenous metastasis by activating downstream NF-κB signaling. Our findings will develop prognosis prediction tools for BLCa patients and discover novel therapeutic biological targets for metastatic BLCa.

Fig. 1. BLACAT3 identification and its relationship with BLCa patient prognosis.

A Flowchart of m6A epitranscriptomic microarray. B Differentially m6A modified lncRNAs (abs(log₂FC) > 0.585, P < 0.05) and differentially expressed lncRNAs ((abs(log₂FC) > 0.585) between paired BLCa and adjacent normal tissues (n = 3). C Volcano plot of differential lncRNA expression profile between 411 BLCa tissues and 19 normal bladder tissues based on TCGA data (abs(log₂FC) > 0.585, P < 0.05). D Venn diagrams of microarray-derived differential lncRNA m6A modification and expression profiles intersected with lncRNA expression profiles from the TCGA dataset. E MeRIP and qRT-PCR were conducted to detect m6A modification levels of BLACAT3 and ENST00000439898.1 (n = 3). F qRT-PCR was used to detect BLACAT3 expression between paired BLCa and adjacent normal tissues (n = 104). G Comparison of BLACAT3 expression between earlier stage (n = 86) and advanced stage (n = 18). H Kaplan-Meier analysis of the relationship between BLACAT3 expression and OS of BLCa patients (Log-rank (Mantel-Cox) test, P < 0.001). 104 BLCa patients were divided into high and low expression groups based on the median value of relative BLACAT3 expression. I Univariate and multivariate regression analyses were conducted to screen the independent predictors associated with OS in BLCa patients. Hazard ratio (HR) and corresponding 95% confidence intervals (CI) are shown. J Nomogram was constructed to predict the prognosis of BLCa patients undergoing RC. Statistical significance was assessed with a two-tailed Student’s t test between two groups, *P < 0.05, **** P < 0.0001.

Fig. 2. Effect of m6A modification on BLACAT3 expression in BLCa.

A MeRIP and qRT-PCR were used to detect the m6A modification levels of BLACAT3 in different BLCa cell lines (RT4, UM-UC-3, 5637, T24, J82 and SW780) and the normal urothelial cell line (SV-HUC-1). B The SRAMP prediction server (https://www.cuilab.cn/sramp) was used to predict potential m6A modification motifs in the BLACAT3 sequence. C Single-base mapping of m6A against adenine in the “GGACU” motif was performed to detect the m6A abundance of BLACAT3 in several BLCa cell lines (UM-UC-3, 5637 and T24). D, E WB were performed to detect the protein level of ALKBH5 in paired MIBC and adjacent normal tissues (n = 12). F, G WB were performed to verify the knockdown efficiency of ALKBH5 in 5637 cells and the overexpression efficiency of ALKBH5 in T24 cells. H Anti-m6A dot blot assay detected the effect of ALKBH5 knockdown or overexpression on the overall m6A modification level of 5637 or T24 cells. I MeRIP and qRT-PCR detected the effect of ALKBH5 knockdown on BLACAT3 m6A enrichment in 5637 cells. J QRT-PCR quantified ALKBH5 knockdown efficiency and BLACAT3 expression in 5637 cells. K QRT-PCR examined the effect of ALKBH5 knockdown on the half-life of BLACAT3 in 5637 cells pretreated with actinomycin-D (5 μg/mL). L MeRIP and qRT-PCR assays were conducted to detect the effect of ALKBH5 overexpression on BLACAT3 m6A enrichment in T24 cells. M QRT-PCR assay was performed to detect the overexpression efficiency of ALKBH5 and BLACAT3 expression in T24 cells. N QRT-PCR assay was performed to examine the effect of ALKBH5 overexpression on the half-life of BLACAT3 in T24 cells pretreated with actinomycin-D (5 μg/mL). ns no significance. Statistical significance was assessed using two-tailed Student’s t test between two groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3. Effects of BLACAT3 on BLCa proliferation in vivo.

A, B In vivo imaging was performed on day 28 after subcutaneous injection of BLACAT3 stable knockdown T24 cells into NSG mice, fluorescent quantitative statistics were performed on representative images. C Tumor volume was measured every 7 days after subcutaneous injection. D, E Anti-Ki67 and anti-CD31 IF staining was conducted to explore the effect of BLACAT3 knockdown on the proliferation and angiogenesis of T24 cells in vivo. Image J software was used for semi-quantitative analysis of representative immunofluorescent images. Scale bars: 20 μm. F, G In vivo imaging was performed on day 28 after subcutaneous injection of 5637 cells with stable BLACAT3 overexpression into NSG mice, and fluorescence quantitative statistics were performed on representative images. H Tumor volume was measured every 7 days after subcutaneous inoculation of 5637 cells. I, J Anti-Ki67 and anti-CD31 immunofluorescent staining were performed to assess the effects of BLACAT3 overexpression on the proliferation and angiogenesis of 5637 cells in vivo. Image J software was used to perform semi-quantitative analyzing of representative fluorescent images. Scale bars: 20 μm. Statistical significance was assessed using two-tailed Student’s t test between two groups, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Effects of BLACAT3 on BLCa angiogenesis and migration in vitro and in vivo.

HUVECs tube formation and transwell migration assay were performed to detect the effects of BLACAT3 knockdown on tumor angiogenesis and migration in T24 (A–C) and 5637 cells (D–F). Scale bars: 2 mm (black lines), 500 μm (orange lines). G, H In vivo imaging was performed on day 28 after tail vein injection of T24 cells with stable BLACAT3 knockdown into NSG mice, and fluorescent quantitative statistics were performed on representative images. I NSG mice were sacrificed on the 28th day after the lung metastasis model was constructed, and the gross lung samples were isolated, fixed, embedded and sliced, and then H&E staining was performed. Scale bars: 30 μm. J–L Anti-Ki67 and anti-CD31 immunofluorescent staining were performed to assess the T24 cell proliferation and angiogenesis in vivo. and fluorescent quantitative statistics were performed on representative pictures. Scale bars: 20 μm. Statistical significance was assessed using two-tailed Student’s t test between two groups, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. NCF2 identification and its interaction with BLACAT3.

A Heatmap showed differentially expressed mRNAs in T24 cells with BLACAT3 knockdown. Abs(log₂(FC)) > 0.585 and P < 0.05 were set as the thresholds. B Gene Ontology (GO) analysis showed that BLACAT3 knockdown was involved in biological functions related to movement and localization. C KEGG enrichment analysis revealed the top 10 signaling pathway regulated by BLACAT3. D Heatmap showed top10 mRNAs down- and up-regulated by BLACAT3 knockdown. E QRT-PCR detected the NCF2 expression between paired BLCa and adjacent normal tissues (n = 104). F Relative expression (Tumor/Normal ratio) of NCF2 between earlier TNM stage group (n = 86) and advanced stage group (n = 18). G Correlation analysis between BLACAT3 and NCF2 expression exhibited a positive correlation, P < 0.0001. H, I Kaplan-Meier survival curves showed the effect of NCF2 expression on the OS of BLCa patients (Log-rank (Mantel-Cox) test, P < 0.001) and DSS of BLCa patients (Log-rank (Mantel-Cox) test, P < 0.05). A total of 104 BLCa patients were divided into high expression group and low expression group by the median of relative NCF2 expression. J NSG mice were subcutaneously injected using T24 cells with BLACAT3 stable knockdown, and the mice were sacrificed on the 28th day. The subcutaneous tumor was fixed, embedded in sections, and then H&E staining and anti-NCF2 IHC staining were performed. K The mice were sacrificed on the 28th day after tail vein injection, and the gross lung samples were isolated, fixed and embedded, and then H&E staining and anti-NCF2 IHC staining were conducted. Scale bars: 1.6 mm (black lines), 40 μm (orange lines). Statistical significance was assessed using two-tailed Student’s t test between two groups, ****P < 0.0001.

Fig. 6. Effects of NCF2 on BLCa angiogenesis and migration in vitro.

A–C WB verified NCF2 knockdown efficiency in T24 and 5637 cells. D–I HUVEC tube formation and transwell migration assays were used to investigate the effects of NCF2 knockdown on pro-angiogenesis and migratory abilities of T24 cells (D–F) and 5637 cells (G–I). Scale bars: 2 mm (black lines), 500 μm (orange lines). J Fluorescence in situ hybridization (FISH) assay detected the subcellular distribution of BLACAT3 in T24 and 5637 cells. 18 S rRNA and U6 were respectively used as cytoplasmic and nuclear internal reference biomarkers. Scale bars: 20 μm. K, L The subcellular distribution of BLACAT3 in T24 and 5637 cells was detected by qRT-PCR assay after isolation of nuclear and cytoplasmic RNAs. GAPDH and U6 were respectively used as cytoplasmic and nuclear internal reference biomarkers. Statistical significance was assessed using two-tailed Student’s t test between two groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. BLACAT3 binds and induces YBX3 to shuttle into the nucleus.

A RNA pull-down and silver staining showed the molecular weight of BLACAT3 binding proteins. B WB assay using the protein sample pulled by BLACAT3 proved that YBX3 is the binding protein of BLACAT3. C, D RNA Immunoprecipitation (RIP) and qRT-PCR assay confirmed that BLACAT3 can bind with YBX3 protein. E, H Western blot demonstrated that BLACAT3 knockdown had no effect on YBX3 protein levels in T24 and 5637 cells. F, I The effect of BLACAT3 knockdown on the subcellular distribution of YBX3 in T24 cells was detected by WB assay after separation of nuclear and cytoplasmic proteins. G, J The effect of BLACAT3 overexpression on YBX3 subcellular distribution in 5637 cells was detected by WB assay after separation of nuclear and cytoplasmic proteins. K NSG mice were subcutaneously injected with T24 cells with BLACAT3 stable knockdown, and the mice were sacrificed on the 28th day. The subcutaneous tumor was isolated, fixed and embedded, and then H&E staining and anti-YBX3 immunofluorescent (IF) staining assessed the effect of BLACAT3 knockdown on the subcellular distribution of YBX3. Scale bars: 1 mm (black and white lines), 20 μm (orange lines). ns no significance. Statistical significance was assessed using two-tailed Student’s t test between two groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. Co-regulation of BLACAT3/YBX3 complex on NCF2 expression by binding NCF2 promoter and enhance NCF2 gene transcription, then activate NF-kB signaling pathway.

Dual-luciferase gene reporter assay (A, C) and ChIP-qPCR assay (B, D) were performed to verify that BLACAT3 can bind YBX3. E ChIP-qPCR was performed to verify BLACAT3/YBX3 complex can bind to NCF2 promoter and regulate the transcription. F, G WB detected the regulation effects of BLACAT3 and/or YBX3 knockdown on NCF2 protein level. H HALLMARK gene set enrichment analysis (GSEA) based on RNA sequencing data revealed typical signaling pathways regulated by BLACAT3. I WB verified potential signaling pathways in T24 and 5637 cells. ns no significance. Statistical significance was assessed using two-tailed Student’s t test between two groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.