Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr;40(15):2635-2650.
doi: 10.1038/s41388-021-01666-z. Epub 2021 Feb 9.

RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Qiuxia Yan  1 , Peng Zeng  2 , Xiuqin Zhou  3 , Xiaoying Zhao  3 , Runqiang Chen  3 , Jing Qiao  3 , Ling Feng  3 , Zhenjie Zhu  3 , Guozhi Zhang  3 , Cairong Chen  4
Affiliations

RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Qiuxia Yan et al. Oncogene. 2021 Apr.

Abstract

The prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Low levels of RBMX expression were associated with poor survival prognoses for Bca patients.
A RBMX mRNA expression levels were decreased in BCa tissues compared to those in the matched adjacent NT tissues from the GSE13507 data set. B Compared to that in the NMIBC tissues (Ta or T1), RBMX mRNA expression levels were decreased in the MIBC tissues (≥T2) in the GSE89 and GSE32548 data sets. C–E Compared to the rates of the patient cohort with low RBMX expression (the lower 25%), the TCGA urothelial cancer patient cohort with high RBMX mRNA expression (the upper 25%) had lower death rates and higher DFS and OS. F, G The patients with highly expressed RBMX identified in the GSE13507 and GSE32548 data sets had longer OS. H, I RBMX RNA levels in the NMIBC tissues (n = 5), MIBC tissues (n = 5), and matched adjacent NT tissues (NT) (n = 10) were detected by qRT-PCR and RT-PCR. J–L RBMX protein levels in the NMIBC tissues (n = 5), MIBC tissues (n = 5), and matched adjacent NT tissues (n = 10) were detected by western blot. Data are presented as the means ± SD.
Fig. 2
Fig. 2. Stable overexpression of RBMX suppressed the malignant phenotypes of the BCa cells in vitro and in vivo.
A, B Stable overexpression of RBMX was measured in 5637 and T24 cells by qRT-PCR and western blot. C, D The Flag-RBMX-pcDNA3.1 plasmid was stably expressed in T24 cells. Flag and RBMX were immunostained using anti-Flag and anti-RBMX antibodies, respectively. E–H The effects of the stable overexpression of RBMX on 5637 and T24 cell growth (E), proliferation (F), colony formation (G), migration, and invasion (H) were determined. I Tumor volume and weight at the end points of subcutaneous xenograft tumors formed by the T24 cells stably transfected with LV-NC or LV-RBMX into nude mice (n = 6 per group). J NOD-SCID mice were treated with via tail vein injection of T24 cells (2 × 106 cells/mouse) stably transfected with LV-NC-Luc or LV-RBMX-Luc. Pulmonary metastasis was detected by an in vivo imaging system (IVIS) (n = 5 per group). K Representative images of the extent of metastasis based on lung observations and H&E staining. L Kaplan–Meier curves of NOD-SCID mice are shown. Data are presented as the means ± SD. M In TCGA urothelial cancer patient cohort, the expression of RBMX was higher in the tissues of no regional lymph node metastasis than metastases in axillary lymph nodes (P = 0.0102).
Fig. 3
Fig. 3. RBMX interacted with the RGG box in hnRNP A1.
A Proteins that interacted with RBMX were identified by silver staining and mass spectrometry. B The RBMX-binding protein was analyzed by g: Profiler (https://biit.cs.ut.ee/gprofiler/). C RBMX-regulated alternative splicing events (A3SS/A5SS, alternative 3′/5′ splice sites; MXE mutually exclusive exons, RI retained introns, SE skipped exons) were detected by RNA-seq of the T24 cells. D PKM pre-mRNA splicing was regulated by RBMX overexpression. E, F The Flag-RBMX or HA-hnRNP A1 plasmids was transfected into HEK293T cells, Flag-RBMX complexes were coimmunoprecipitated by anti-Flag antibody, and then hnRNP A1 was detected by anti-hnRNP A1 antibody (E); HA-hnRNP A1 complexes were coimmunoprecipitated by anti-HA antibody and then detected by anti-RBMX antibody (F). G RBMX (red) and hnRNP A1 (green) colocalization in the T24 cells was detected by confocal microscopy. H Diagram of the wild-type hnRNP A1 and mutation constructs with different domains. I, J Wild-type hnRNP A1 and different mutation constructs together with the Flag-RBMX plasmid were transfected into HEK293T cells, anti-HA antibody was used for the coimmunoprecipitation, and Flag-RBMX was detected using anti-Flag antibody (I); anti-Flag antibody was used to coimmunoprecipitate, and wild-type hnRNP A1 and different mutation constructs were detected using anti-HA antibody (J). K, L Wild-type hnRNP A1 and RGGMUT plasmids together with the Flag-RBMX plasmid were co-transfected into HEK293T cells; the interactions of RBMX with hnRNP A1 RGGMUT were determined as described in (I)(K) and (J)(L).
Fig. 4
Fig. 4. RBMX antagonized the hnRNP A1-induced aggressive phenotype of the BCa cells.
Flag-RBMX, HA-hnRNP A1, or Flag-RBMX plasmid together with the HA-hnRNP A1 plasmid were transfected into 5637 and T24 cells, and the indicated protein expression (A), cell growth (B), colony formation (C), migration, and invasion (D) were determined. Data are presented as the means ± SD.
Fig. 5
Fig. 5. RBMX hindered the binding of the RGG box in hnRNP A1 to the sequences flanking exon 9 of PKM.
A The Flag-RBMX plasmid was transfected into T24 cells, and the protein levels of RBMX, hnRNP A1, PKM1, and PKM2 were detected. B T24 cell nuclear extracts were affinity-purified using the indicated biotin-labeled RNAs, and then the eluted proteins were indicated by anti-RBMX and anti-hnRNP A1 antibodies. C Flag-RBMX plasmid at the specified doses was transfected into T24 cells, and the hnRNP A1 expression level was detected after affinity purification using biotin-labeled RNA EI9 (50–68). D Wild-type hnRNP A1 and MUT1–MUT4 were transfected into T24 cells, and the indicated protein levels were determined after affinity purification using biotin-labeled RNA EI9 (50–68). E Wild-type hnRNP A1 or hnRNP A1 MUT3–MUT4 plasmids together with the Flag-RBMX plasmid were transfected into T24 cells, and the indicated protein levels were measured after affinity purification using biotin-labeled RNA EI9 (50–68). F Wild-type hnRNP A1 or RGGMUT was transfected into T24 cells, and the indicated protein levels were determined after affinity purification using biotin-labeled RNA EI9 (50–68).
Fig. 6
Fig. 6. RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM splicing in the T24 cells.
A, B Flag-RBMX plasmids (A) or the anti-RBMX siRNAs (B) were transfected into T24 cells, and PKM splicing was performed using PstI. C Flag-RBMX, HA-hnRNP A1, or Flag-RBMX plasmid together with the HA-hnRNP A1 plasmid were transfected into T24 cells, and then, PKM splicing was induced using PstI. D PKM splicing was performed in the NMIBC tissues, MIBC tissues, and matched adjacent normal tissues. E The mRNA levels of PKM1 and PKM2 were positively and negatively correlated with the RBMX mRNA levels in the NMIBC tissues (n = 3), MIBC tissues (n = 3), and matched adjacent normal tissues (n = 6). F–K Flag-RBMX plasmid (F, G), RBMX siRNAs (H, I), or hnRNP A1 siRNAs (J, K) were transfected into T24 cells, and then, the glucose uptake and lactate production were measured. L, M Flag-RBMX, HA-hnRNP A1, or Flag-RBMX together with the HA-hnRNP A1 plasmid were transfected into T24 cells, and then, the glucose uptake and lactate production were measured. N, O Flag-RBMX, HA-PKM2, or Flag-RBMX plasmid together with the HA-PKM2 plasmid were transfected into T24 cells, and then, the glucose uptake and lactate production were measured. Data are presented as the means ± SD.
Fig. 7
Fig. 7. RBMX counteracted the PKM2 overexpression-induced aggressive phenotype.
A–D Flag-RBMX, HA-PKM2, or Flag-RBMX plasmid together with the HA-PKM2 plasmid were transfected into 5637 and T24 cells, and the indicated protein expression (A), cell growth (B), colony formation (C), and migration and invasion (D) were detected. E A schematic model of the mechanism underlying the role of RBMX in BCa aerobic glycolysis through regulating PKM alternative splicing mediated by hnRNP A1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. doi: 10.3322/caac.21492. - DOI - PubMed
    1. Babjuk M, Burger M, Comperat EM, Gontero P, Mostafid AH, Palou J, et al. European Association of Urology Guidelines on Non-muscle-invasive Bladder Cancer (TaT1 and Carcinoma In Situ) - 2019 Update. Eur Urol. 2019;76:639–57. - PubMed
    1. Cambier S, Sylvester RJ, Collette L, Gontero P, Brausi MA, van Andel G, et al. EORTC nomograms and risk groups for predicting recurrence, progression, and disease-specific and overall survival in non-muscle-invasive stage Ta-T1 urothelial bladder cancer patients treated with 1-3 years of maintenance bacillus calmette-guerin. Eur Urol. 2016;69:60–9. - PubMed
    1. Ploussard G, Shariat SF, Dragomir A, Kluth LA, Xylinas E, Masson-Lecomte A, et al. Conditional survival after radical cystectomy for bladder cancer: evidence for a patient changing risk profile over time. Eur Urol. 2014;66:361–70. - PubMed
    1. Vishnu P, Mathew J, Tan WW. Current therapeutic strategies for invasive and metastatic bladder cancer. Onco Targets Ther. 2011;4:97–113. - PMC - PubMed

Publication types

MeSH terms

Substances