Oncogenic SLC2A11-MIF fusion protein interacts with polypyrimidine tract binding protein 1 to facilitate bladder cancer proliferation and metastasis by regulating mRNA stability

Abstract

Chimeric RNAs, distinct from DNA gene fusions, have emerged as promising therapeutic targets with diverse functions in cancer treatment. However, the functional significance and therapeutic potential of most chimeric RNAs remain unclear. Here we identify a novel fusion transcript of solute carrier family 2-member 11 (SLC2A11) and macrophage migration inhibitory factor (MIF). In this study, we investigated the upregulation of SLC2A11-MIF in The Cancer Genome Atlas cohort and a cohort of patients from Sun Yat-Sen Memorial Hospital. Subsequently, functional investigations demonstrated that SLC2A11-MIF enhanced the proliferation, antiapoptotic effects, and metastasis of bladder cancer cells in vitro and in vivo. Mechanistically, the fusion protein encoded by SLC2A11-MIF interacted with polypyrimidine tract binding protein 1 (PTBP1) and regulated the mRNA half-lives of Polo Like Kinase 1, Roundabout guidance receptor 1, and phosphoinositide-3-kinase regulatory subunit 3 in BCa cells. Moreover, PTBP1 knockdown abolished the enhanced impact of SLC2A11-MIF on biological function and mRNA stability. Furthermore, the expression of SLC2A11-MIF mRNA is regulated by CCCTC-binding factor and stabilized through RNA N4-acetylcytidine modification facilitated by N-acetyltransferase 10. Overall, our findings revealed a significant fusion protein orchestrated by the SLC2A11-MIF-PTBP1 axis that governs mRNA stability during the multistep progression of bladder cancer.

Keywords: PTBP1; SLC2A11–MIF; bladder cancer; fusion protein; mRNA stability; metastasis.

FIGURE 1

Characterization of the chimeric RNA SLC2A11–MIF and its parental genes in bladder cancer. (A) Mechanisms of chimeric RNA SLC2A11–MIF formation. Red or blue segments represent exons, and gray segments represent introns or intergenic regions. Cis‐splicing occurs between adjacent genes. Transcriptional readthrough occurs between SLC2A11 and MIF on the same strand with identical transcriptional orientation. (B) Sanger sequencing results of SLC2A11–MIF, with the junction indicated by a red dashed line. (C) SLC2A11–MIF mRNA expression in human uroepithelial cells, SV‐HUC‐1 cells, and 4 bladder cancer cell lines, UM‐UC‐3, T24, 5637, and TCCSUP cells. (D) The gene counts of SLC2A11–MIF in NATs and BCa tissues were analyzed using data from the TCGA database. (E) The expression of SLC2A11–MIF was detected in bladder cancer tissues paired with NATs in the SYSMH cohort. (F) The expression of parental genes in BCa tissues paired with normal adjacent normal tissues in the TCGA cohort was detected. (G) The expression of parental genes was measured by qRT‐PCR in 94 BCa tissues paired with NATs. (H and I) Kaplan–Meier curves for OS and DFS of patients with bladder cancer with high versus low expression of parental genes. *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates not statistically significant.

FIGURE 2

Knockdown of SLC2A11–MIF inhibited proliferation in vitro and tumor growth in vivo. (A) qRT‐PCR analysis was conducted to evaluate the expression of SLC2A11–MIF in SLC2A11–MIF‐silenced and control BCa cells. (B) Cell viability determined by a CCK‐8 assay was evaluated in T24 and UM‐UC‐3 cells with SLC2A11–MIF knockdown. (C) Colony formation assays were performed in T24 and UM‐UC‐3 cells with SLC2A11–MIF knockdown. (D) Quantification of cell apoptosis in T24 and UM‐UC‐3 cells with knockdown of SLC2A11–MIF. (E) The growth of UM‐UC‐3 tumors with SLC2A11–MIF knockdown was assessed every 3 days, and tumor growth curves were generated. The mean and standard deviation (SD) of the tumor volumes measured in six mice are shown. (F) Representative image of subcutaneous tumors in which SLC2A11–MIF knockdown is presented. (G) Weight (mg) of tumors with SLC2A11–MIF knockdown after surgical dissection. (H) Representative images of H&E and IHC staining demonstrating Ki67 expression in tumors. Scale bars: 100 µm (black). (I) Histogram of the H‐scores of Ki67 cells with SLC2A11–MIF knockdown. (J and K) Representative images and histograms showing the proportions of TUNEL‐positive cells. Scale bars: 50 µm (white). **p < 0.01, ***p < 0.001.

FIGURE 3

Knockdown of SLC2A11–MIF suppressed the metastatic behavior of bladder cancer cells in vitro and lymphatic metastasis in vivo. (A) Representative images and histograms of wound healing assays in T24 and UM‐UC‐3 cells demonstrating decreased cellular motility following knockdown of SLC2A11–MIF. (B and C) Representative images and histograms of migration assays in T24 and UM‐UC‐3 cells reveal decreased cell migratory capacity after knockdown of SLC2A11–MIF. (D and E) Representative images and histograms illustrating the invasion of T24 and UM‐UC‐3 cells after silencing SLC2A11–MIF. (F) Representative images showing tumor invasion into the surrounding muscle in the SLC2A11–MIF knockdown groups compared with the respective controls. M represents muscle, while T represents tumor. Arrows indicate invasive tissues. The histogram shows the percentage of tumor invasion into the surrounding muscle between the SLC2A11–MIF‐knockdown group and the control group. (G and H) Representative images of dissected popliteal LNs and histogram analysis of the LN volume. (I) Representative images of H&E and IHC staining for LN status. Scale bars, 500 mm (black) and 50 mm (red). **p < 0.01, ***p < 0.001.

FIGURE 4

Characterization and functional analysis of the SLC2A11–MIF fusion protein. (A) The flowchart illustrates the comprehensive analysis of full‐length SLC2A11–MIF. (B) Structures of the two isoforms of the SLC2A11–MIF fusion are depicted, with blocks representing exons and lines representing introns or the intergenic region. (C) Top: Diagram showing the variants of SLC2A11–MIF mRNA and the primers used for RT‐PCR detection of exon 4 (primer E4), exons 5 (primer E5), exons 6 and 7 (primer E6‐7), exons 8 (primer E8), and exons 9 (primer E9). Bottom: Expression levels of S‐M‐L and S‐M‐S isoforms were assessed using RT‐PCR in UM‐UC‐3 cells. (D) Representative images demonstrating the presence of S‐M‐L and S‐M‐S by RT‐PCR in BCa tissues and metastatic lymph node (LN+) tissues. (E) Western blotting was performed to determine S‐M‐S‐FLAG expression levels in S‐M‐S‐overexpressing cells and control cells. (F) Cell viability of S‐M‐S‐overexpressing T24 and UM‐UC‐3 cells. (G) Colony formation in S‐M‐S‐overexpressing T24 and UM‐UC‐3 cells. (H and I) The percentages (%) of cell populations at different stages of the cell cycle are provided for SLC2A11–MIF‐overexpressing T24 and UM‐UC‐3 cells. (J) Representative images and histograms of migration assays in T24 and UM‐UC‐3 cells reveal increased cell migratory capacity after overexpressing S‐M‐S. Scale bars: black, 100 µm. (K) Representative images and histograms illustrating the invasion of T24 and UM‐UC‐3 cells overexpressing S‐M‐S. Scale bars: black, 100 µm. (L) Histograms from wound healing assays demonstrating cellular motility following overexpression of S‐M‐S in T24 and UM‐UC‐3 cells. Scale bars: black, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

SLC2A11–MIF enhanced the proliferation and metastasis of BCa cells in vivo. (A) Growth curves of UM‐UC‐3 tumors with control or SLC2A11–MIF overexpression. (B) Representative images of subcutaneous tumors with control or SLC2A11–MIF overexpression are presented. (C) The weight (mg) of tumors with control or SLC2A11–MIF overexpression was measured after surgical dissection. (D) Representative images and a histogram of H&E and IHC staining images demonstrating Ki67 expression in tumors. Scale bars: 100 µm (black). (E) Representative images demonstrating tumor invasion into the surrounding muscle in the SLC2A11–MIF‐overexpressing group compared with the corresponding control group. The letter M denotes muscle, while T represents tumor. Arrows indicate areas of tissue invasion. (F) The histogram illustrates the percentage of tumor infiltration into the surrounding muscle between the SLC2A11–MIF‐overexpressing group and the control group. (G and H) Representative images of dissected popliteal lymph nodes and histogram analysis were performed to assess the volume of the lymph nodes. (I) LN status percentages in all groups (n = 6 per group). (J) Kaplan‒Meier survival analysis of mice inoculated with SLC2A11–MIF‐overexpressing or control cells. (K) Representative images of H&E and IHC staining were used to confirm the status of the lymph nodes (n = 6). Scale bars, 500 µm (black) and 50 µm (red). **p < 0.01.

FIGURE 6

The fusion protein SLC2A11–MIF directly interacts with PTBP1 to play key roles in BCa. (A) Co‐immunoprecipitation (Co‐IP) was conducted in S‐M‐S‐FLAG‐overexpressing T24 cells using an anti‐FLAG or negative control IgG antibody, followed by silver staining. The red arrows indicate the localization of PTBP1 (above) and SLC2A11–MIF (below) protein bands. (B) Co‐IP and western blotting analysis revealed the interaction between SLC2A11–MIF and PTBP1. (C) Cell viability was evaluated in SLC2A11–MIF‐overexpressing or control cells with PTBP1 knockdown in T24 and UM‐UC‐3 cells. (D and E) Colony formation was assessed in SLC2A11–MIF‐overexpressing or control cells in combination with PTBP1 knockdown. (F and G) Representative images and histograms of migration were evaluated using SLC2A11–MIF‐overexpressing or control cells with PTBP1 knockdown. Scale bars: black, 100 µm. (H and I) Representative images and histograms of invasion were evaluated using SLC2A11–MIF‐overexpressing or control cells with PTBP1 knockdown. Scale bars: black, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

SLC2A11–MIF modulates the stability of the PLK1, ROBO1, and PIK3R3 mRNAs in a PTBP1‐mediated manner. (A) A heatmap depicting mRNA expression in T24 and UM‐UC‐3 cells transfected with control or SLC2A11–MIF siRNA. (B) Differentially expressed genes identified from the microarray were validated by qRT‐PCR in T24 and UM‐UC‐3 cells. (C) Western blotting was performed to assess the expression of SLC2A11–MIF target genes, with GAPDH serving as the internal control. (D) Pearson correlations between the expression levels of SLC2A11–MIF and PLK1, ROBO1, and PIK3R3 were determined by qRT‐PCR analysis of samples from 50 patients with bladder cancer. (E and F) mRNA and protein expression of SLC2A11–MIF target genes was evaluated in cells overexpressing SLC2A11–MIF or in control cells with PTBP1 knockdown. (G) UM‐UC‐3 cells transfected with control or SLC2A11–MIF siRNA were treated with actinomycin D (5 mg/mL) for the indicated durations. (H) UM‐UC‐3 cells with stable control expression, SLC2A11–MIF overexpression, or SLC2A11–MIF overexpression with PTBP1 siRNA were treated with actinomycin D (5 mg/mL) for the indicated time periods. Total RNA was purified and analyzed using qRT‐PCR to determine the mRNA half‐lives of PLK1, ROBO1, and PIK3R3. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

SLC2A11–MIF is a product of cis‐SAGe, and its stability is mediated by NAT10. (A) Top: Schematic showing cis‐splicing between adjacent genes (cis‐SAGe). Blocks represent exons, while lines represent introns or intergenic regions. The arrowhead indicates the oligo primer used for reverse transcription. The primers E8‐F and I8‐R anneal to exon 8 and intron 8 of SLC2A11, respectively. Bottom: RNA from the bladder cancer cell lines T24 and UM‐UC‐3, as well as from the human uroepithelial cell line SV‐HUC‐1, was initially treated with DNase I. Subsequently, the cells were divided into two groups: one with the avian myeloblastosis virus reverse transcriptase (AMV‐RT) enzyme and the other without it. The correct product was observed exclusively in samples containing the AMV‐RT enzyme. (B) Sanger sequencing confirmed the validity of the PCR products. (C) qRT‐PCR analysis was performed to evaluate the expression levels of SLC2A11–MIF in CTCF‐silenced cells and control cells. (D) The relative expression of SLC2A11–MIF was measured by qRT‐PCR after NAT10 silencing. (E) Pearson correlations between the expression levels of SLC2A11–MIF and NAT10 were determined by qRT‐PCR analysis using samples from 50 bladder cancer patients. (F) Change in SLC2A11–MIF mRNA stability was assessed by qRT‐PCR following actinomycin D treatment after NAT10 knockdown. (G) RIP assays were conducted on UM‐UC‐3 cells to analyze the enrichment of SLC2A11–MIF mRNA relative to the nontargeting IgG control using qRT‐PCR analysis. (H) Proposed model of the interaction of the SLC2A11–MIF fusion protein with PTBP1 to promote the proliferation and metastasis of bladder cancer cells via the regulation of mRNA stability. The image was created using BioRender.com. *p < 0.05, **p < 0.01, ***p < 0.001.