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Identify Potential Urine Biomarkers for Bladder Cancer Prognosis Using NGS Data Analysis and Experimental Validation

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Abstract

Bladder cancer (BC) is one of the most often reported malignancies globally, with a high recurrence rate and associated morbidity and mortality, especially in advanced BC. There has been a surge in the number of molecular targets revealed for BC prognosis and treatment. However, there is still a great need to discover novel biomarkers. Consequently, the current study investigated biomarkers that might indicate the progression of bladder cancer. In this study, bioinformatics analysis was done on a single GEO dataset, and TCGA-BLCA information was connected with differentially expressed genes (DEGs). The levels of mRNA and protein expression were validated using qRT-PCR. According to our findings, CRYAB, ECM1, ALDOB, AOC, GPX3, IGFBP7, AQP2, LASS2, TMEM176A, GALNT1, and LASS2 were highly enriched in cell division, identical protein binding, and developmental process in bladder cancer patients. In addition, among the highly differentiated genes, ECM1, GALNT1, LASS2, and GPX3 showed significant molecular alterations in BC, which are crucial for marker identification. Moreover, the mRNA, CNVs, and protein levels of ECM1, GALNT1, LASS2, and GPX3 were significantly increased in BC patients. Our predictions and analysis studies stated that these four genes act as urine biomarkers and played a crucial role in disease prognosis and the therapeutic process of bladder cancer. Our outcomes showed that these four novel urine biomarkers have the potential to provide innovative diagnostics, early predictions, and disease targets, ultimately improving the BC patient’s prognosis.

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Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ALDOB :

Aldolase B enzyme

AOC :

Allene oxide cyclase

AQP2 :

Aquaporin 2

BC :

Bladder cancer

cDNA :

Complementary deoxyribonucleic acid

CNVs :

Copy number variation

CRYAB :

Alpha-crystallin B chain

DEG :

Differentially expressed genes

ECM1 :

Extracellular matrix protein 1

EV :

Extracellular vesicles

GalNAc :

N-acetyl-d-galactosamine

GALNT1 :

N-acetylgalactosaminyltransferase 1

GEO :

Gene Expression Omnibus

GPX3 :

Glutathione peroxidase 3

IGFBP7 :

Insulin-like growth factor-binding protein 7

KEGG :

Kyoto Encyclopedia of Genes and Genomes

LASS2 :

LAG1 longevity-assurance homolog 2

MIBC :

Muscle-invasive bladder cancer

mRNA :

Messenger RNA

NGS :

Next-generation sequencing

NIMBC :

Non-muscle-invasive bladder carcinomas

NMP22 :

Nuclear matrix protein 22

PPI :

Protein-protein interaction networks

qRT-PCR :

Real-time quantitative reverse transcription-polymerase chain reaction

STRING :

Search Tool for the Retrieval of Interacting Genes/Proteins

TCGA :

The Cancer Genome Atlas

TCGA-BLCA :

Cancer genome atlas bladder cancer

TMEM176A :

Human transmembrane protein 176A

UALCAN :

User-friendly, interactive web resource for analyzing cancer transcriptome data

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Author information

Authors and Affiliations

  1. Department of Urology, Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, 030032, China

    Ruimin Ren, Huang Wang & Liulei Xie

  2. AMR and Nanomedicine Lab, Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, 600077, India

    Saravanan Muthupandian

  3. Department of Urology, First Hospital of Shanxi Medical University, Taiyuan, 030000, China

    Xiaofeng Yang

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  4. Saravanan Muthupandian
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Contributions

RR: funding acquisition, in silico and in vivo experiments, software, data analysis. HW: investigation, data curation, formal analysis. LX: methodology, project administration; SM: conceptualization, editing the manuscript. XY: funding, project administration, supervision drafts the manuscript.

Corresponding author

Correspondence to Xiaofeng Yang.

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Ethical Approval

This study involved animals and ethics approved by the Department of Urology, Third Hospital of Shanxi Medical University Hospital, China, with ethics number YXLL-2021-066.

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Figure S1

Tissue specificity of differentially expressed genes. A) GALNT1, B) ECM1, C) CERS2 and D) GPX3. (PNG 152 kb)

High resolution image (TIF 6963 kb)

Figure S2

Normalized expression of selected differential marker genes (CRYAB, ALDOB, IGFBP7, RHCG and TMEM176A) in Bladder cancer. A) mRNA expression pattern of CRYAB, ALDOB, IGFBP7, RHCG and TMEM176A marker genes (as a box-whisker plot) in normal and primary tumor samples. B) Expression pattern of the top marker genes based on tumor subgroups based on race. (PNG 49 kb)

High resolution image (TIF 6399 kb)

Figure S3

A) Molecular alterations for top differential expressed genes. B) Copy number variations of the DEGs. (PNG 93 kb)

High resolution image (TIF 7262 kb)

Figure S4

mRNA expression profile in bladder cancer and other cancer types. Pan cancer gene expression profile of A) CRYAB, B) ALDOB, C) IGFBP7, and D) TMEM176A. (PNG 69 kb)

High resolution image (TIF 6413 kb)

Figure S5

Epigenetic analysis of marker genes in BLCA. A) Methylation pattern of the top marker genes (CRYAB, IGFBP7, ALDOB and TMEM176A) in normal and primary tumor samples. B) Expression pattern of the CRYAB, IGFBP7, ALDOB and TMEM176A marker genes based on tumor subgroups based on race. (PNG 46 kb)

High resolution image (TIF 6389 kb)

Figure S6

Survival analysis of marker genes in BLCA. A) The Kaplan–Meier plotters based on bladder cancer datasets showed patients with high CRYAB expression have poor overall survival, B) IGFBP7, C) ALDOB and D) TMEM176A. (PNG 47 kb)

High resolution image (TIF 6320 kb)

Table S1

Supplementary Material S1 (3.19 MB xls)

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Ren, R., Wang, H., Xie, L. et al. Identify Potential Urine Biomarkers for Bladder Cancer Prognosis Using NGS Data Analysis and Experimental Validation. Appl Biochem Biotechnol 195, 2947–2964 (2023). https://doi.org/10.1007/s12010-022-04234-7

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