Revealing the function and mechanism of piRNA-related genes in bladder cancer through single-cell sequencing and methylation analyses and construction of prognostic features based on consensus clustering

Abstract

Background: This study aimed to explore the functions and potential mechanisms of PIWI-interacting RNA-related genes (piRPGs) in bladder cancer (BC) development and to identify potential prognostic genes.

Methods: This study used differential analysis and machine learning techniques to identify the differentially expressed piRPGs in BC. Consensus clustering was performed on The Cancer Genome Atlas-Urothelial Bladder Carcinoma dataset, and univariate and multivariate analyses were conducted to construct a BC prognostic model consisting of 3 piRPGs. Kaplan-Meier survival curves were used for survival analysis. Quantitative polymerase chain reaction was performed to validate the expression levels of piRPGs in BC cells and tissues. The functional roles and potential mechanisms of piRPGs in BC were investigated via single-cell sequencing and differentially methylated position sequencing. The DSigDB and CellMiner databases were used to screen for small-molecule drugs associated with piRPGs.

Results: This study identified 6 piRPGs that were significantly associated with BC: MAPK13, INHBA, LAMB2, DDX3X, TARBP2, and CDK2. A prognostic model comprising MAPK13, INHBA, and LAMB2 was constructed using consensus clustering technology. Kaplan-Meier curves demonstrated significantly prolonged survival in cluster 2 compared with cluster 1 (p < 0.01), validating the effectiveness of the prognostic model. Single-cell sequencing confirmed that MAPK13 expression was significantly upregulated in bladder tissues (p < 0.001). Methylation site sequencing and methylation-specific polymerase chain reaction revealed significantly decreased methylation levels of INHBA and MAPK13 in BC tissues, which were inversely correlated with their expression levels.

Conclusions: This study effectively developed a 3-gene prognostic signature comprising MAPK13, INHBA, and LAMB2 using consensus clustering and multifactorial logistic regression. In addition, the functional roles and intrinsic mechanisms of piRPGs in bladder carcinogenesis were comprehensively explored using single-cell sequencing, methylation sequencing, and functional enrichment analysis.

Keywords

PIWI-interacting RNA-related gene / DNA methylation / Bladder cancer / MAPK13 / INHBA

Acknowledgments

None.

Statement of ethics

This study was approved by the Ethics Committee of the Clinical Research Institution of Shandong University Provincial Hospital (authorization number: SWYX: NO. 2023-454), and all experimental procedures were conducted in accordance with the principles outlined in the Declaration of Helsinki.Written informed consent was obtained from all patients after they were fully informed of the study objectives, procedures, potential risks, and benefits.

Conflict of interest statement

JL is an Associate Editor, FW is a member of Editorial Board of Current Urology. They confirm no involvement in any stage of this article’s review process, ensuring unbiased editorial decision-making. The other authors declare no conflicts of interest.

Funding source

This work was supported by the Shandong Provincial Nature Science Foundation (ZR2020QH240).

Author contributions

XS was responsible for the experiments and manuscript writing;

DJ contributed to the bioinformatics analysis;

XS performed the data analysis;

YJ prepared part of the figures;

FW and HN were in charge of sample collection;

JL reviewed and revised the entire manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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