Comprehensive Urinary Proteome Profiling Analysis Identifies Diagnosis and Relapse Surveillance Biomarkers for Bladder Cancer.

Bladder cancer (BCa) is the predominant malignancy of the urinary system. Herein, a comprehensive urine proteomic feature was initially established for the noninvasive diagnosis and recurrence monitoring of bladder cancer. 279 cases (63 primary BCa, 87 nontumor controls (NT), 73 relapsed BCa (BCR), and 56 nonrelapsed BCa (BCNR)) were collected to screen urinary protein biomarkers. 4761 and 3668 proteins were qualified and quantified by DDA and sequential window acquisition of all theoretical mass spectra (SWATH-MS) analysis in two discovery sets, respectively. Upregulated proteins were validated by multiple reaction monitoring (MRM) in two independent combined sets. Using the multi-support vector machine-recursive feature elimination (mSVM-RFE) algorithm, a model comprising 13 proteins exhibited good performance between BCa and NT with an AUC of 0.821 (95% CI: 0.675-0.967), 90.9% sensitivity (95% CI: 72.7-100%), and 73.3% specificity (95% CI: 53.3-93.3%) in the diagnosis test set. Meanwhile, an 11-marker classifier significantly distinguished BCR from BCNR with 75.0% sensitivity (95% CI: 50.0-100%), 81.8% specificity (95% CI: 54.5-100%), and an AUC of 0.784 (95% CI: 0.609-0.959) in the test cohort for relapse surveillance. Notably, six proteins (SPR, AK1, CD2AP, ADGRF1, GMPS, and C8A) of 24 markers were newly reported. This paper reveals novel urinary protein biomarkers for BCa and offers new theoretical insights into the pathogenesis of bladder cancer (data identifier PXD044896).

Transcriptome-wide 1-methyladenosine functional profiling of messenger RNA and long non-coding RNA in bladder cancer.

Introduction: Post-transcriptional RNA modifications are crucial regulators of tumor development and progression. In many biological processes, N1-methyladenosine (m1A) plays a key role. However, little is known about the links between chemical modifications of messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) and their function in bladder cancer (BLCA). Methods: Methylated RNA immunoprecipitation sequencing and RNA sequencing were performed to profile mRNA and lncRNA m1A methylation and expression in BLCA cells, with or without stable knockdown of the m1A methyltransferase tRNA methyltransferase 61A (TRMT61A). Results: The analysis of differentially methylated gene sites identified 16,941 peaks, 6,698 mRNAs, and 10,243 lncRNAs in the two groups. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the differentially methylated and expressed transcripts showed that m1A-regulated transcripts were mainly related to protein binding and signaling pathways in cancer. In addition, the differentially genes were identified that were also differentially m1A-modified and identified 14 mRNAs and 19 lncRNAs. Next, these mRNAs and lncRNAs were used to construct a lncRNA-microRNA-mRNA competing endogenous RNA network, which included 118 miRNAs, 15 lncRNAs, and 8 mRNAs. Finally, the m1A-modified transcripts, SCN2B and ENST00000536140, which are highly expressed in BLCA tissues, were associated with decreased overall patient survival. Discussion: This study revealed substantially different amounts and distributions of m1A in BLCA after TRMT61A knockdown and predicted cellular functions in which m1A may be involved, providing evidence that implicates m1A mRNA and lncRNA epitranscriptomic regulation in BLCA tumorigenesis and progression.

Discovery and validation of bladder cancer related excreted nucleosides biomarkers by dilution approach in cell culture supernatant and urine using UHPLC-MS/MS.

The exploration of nucleoside changes in human biofluids has profound potential for cancer diagnosis. Herein, we developed a rapid methodology to quantify 17 nucleosides by UHPLC-MS/MS. Five pairs of isomers were successfully separated within 8 min. The ME was mostly eliminated by sample dilution folds of 1000 for urine and 40 for CCS. The optimized method was firstly applied to screen potential nucleoside biomarkers in CCS by comprising bladder cancer cell lines (5637 and T24) and normal human bladder cell line SV-HUC-1 together with student's t-test and OPLS-DA. Nucleosides with significant differences in the supernatant of urine samples were also uncovered comparing BCa with the non-tumor group, as well as a comparison of BCa recurrence group with the non-recurrence group. By intersecting the differential nucleosides in CCS and urine supernatant, and then further confirmed using validation sets, the combination of m3C and m1A with AUC of 0.775 was considered as a potential biomarker for bladder cancer diagnosis. A panel of m3C, m1A, m1G, and m22G was defined as potential biomarkers for bladder cancer prognosis with an AUC of 0.819. Above all, this method provided a new perspective for diagnosis and recurrence monitoring of bladder cancer. SIGNIFICANCE: The exploration of nucleoside changes in body fluids has profound potential for the diagnosis and elucidation of the pathogenesis of cancer. In this study, we developed a rapid methodology for the simultaneous quantitative determination of 17 nucleosides in the supernatant of cells and urine samples using UHPLC-MS/MS to discover and validate bladder cancer related excreted nucleoside biomarkers. The results of this paper provide a new strategy for diagnosis and postoperative recurrence monitoring of bladder cancer and provide theoretical support for the exploration of its pathogenesis.

Long Noncoding RNA HAND2-AS1 Suppresses Cell Proliferation, Migration, and Invasion of Bladder Cancer via miR-17-5p/KLF9 Axis.

Bladder cancer (BC) is the most common type of malignant tumor in the genitourinary system. Through the microarray analysis of clinical samples, long noncoding RNA HAND2-AS1 expression was found to be downregulated in BC tissues. However, the function of HAND2-AS1 on BC and underlying mechanism are unclear. In this study, the correlations of HAND2-AS1 with clinicopathological parameters in BC patients were determined. The gain- and loss-of-function experiments were conducted to examine the role of HAND2-AS1 in malignant behaviors of BC cells in vitro and in vivo. Then, we paid attention to miR-17-5p/KLF9 axis to illustrate the molecular mechanism. Results showed that HAND2-AS1 was downregulated in BC tissues, and its overexpression significantly inhibited cell proliferation, migration, and invasion in vitro, as well as tumor growth in vivo. Knockdown of HAND2-AS1 caused an opposite effect on BC cell malignancies. Furthermore, miR-17-5p was shown to be a direct target of HAND2-AS1, and it reversed the inhibitory effect of HAND2-AS1 on BC malignancies. Also, as a downstream factor of miR-17-5p, KLF9 silencing was demonstrated to mediate the role of miR-17-5p inhibitor in BC cell proliferation and invasion. Thus, it suggests that HAND2-AS1 acts as a suppressor in BC development through miR-17-5p/KLF9 axis.

A Novel CpG Methylation Risk Indicator for Predicting Prognosis in Bladder Cancer.

PURPOSE: Bladder cancer (BLCA) is one of the most common cancers worldwide. In a large proportion of BLCA patients, disease recurs and/or progress after resection, which remains a major clinical issue in BLCA management. Therefore, it is vital to identify prognostic biomarkers for treatment stratification. We investigated the efficiency of CpG methylation for the potential to be a prognostic biomarker for patients with BLCA. PATIENTS AND METHODS: Overall, 357 BLCA patients from The Cancer Genome Atlas (TCGA) were randomly separated into the training and internal validation cohorts. Least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) were used to select candidate CpGs and build the methylation risk score model, which was validated for its prognostic value in the validation cohort by Kaplan-Meier analysis. Hazard curves were generated to reveal the risk nodes throughout the follow-up. Gene Set Enrichment Analysis (GSEA) was used to reveal the potential biological pathways associated with the methylation model. Quantitative real-time polymerase chain reaction (PCR) and western blotting were performed to verify the expression level of the methylated genes. RESULTS: After incorporating the CpGs obtained by the two algorithms, CpG methylation of eight genes corresponding to TNFAIP8L3, KRTDAP, APC, ZC3H3, COL9A2, SLCO4A1, POU3F3, and ADARB2 were prominent candidate predictors in establishing a methylation risk score for BLCA (MRSB), which was used to divide the patients into high- and low-risk progression groups (p < 0.001). The effectiveness of the MRSB was validated in the internal cohort (p < 0.001). In the MRSB high-risk group, the hazard curve exhibited an initial wide, high peak within 10 months after treatment, whereas some gentle peaks around 2 years were noted. Furthermore, a nomogram comprising MRSB, age, sex, and tumor clinical stage was developed to predict the individual progression risk, and it performed well. Survival analysis implicated the effectiveness of MRSB, which remains significant in all the subgroup analysis based on the clinical features. A functional analysis of MRSB and the corresponding genes revealed potential pathways affecting tumor progression. Validation of quantitative real-time PCR and western blotting revealed that TNFAIP8L3 was upregulated in the BLCA tissues. CONCLUSION: We developed the MRSB, an eight-gene-based methylation signature, which has great potential to be used to predict the post-surgery progression risk of BLCA.