The value of urinary exosomal microRNA-21 in the early diagnosis and prognosis of bladder cancer

Abstract

Bladder cancer (BC) poses high morbidity and mortality, with urinary exosomal microRNA (miR)-21 showing potential value in its diagnosis and prognosis, and we probed its specific role. We prospectively selected 116 BC patients and 116 healthy volunteers as the BC and control groups, respectively. BC urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 relative expression levels were assessed. The correlations between clinical indexes and urinary exosomal miR-21, prognostic value of miR-21, and diagnostic value of the five candidate miRNAs, urine cytology, and miRNA joint diagnostic panel for BC and urinary exosomal miR-21, miR-4454, and urine cytology for Ta-T1 and T2-T4 stage BC were analyzed. Urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 were highly expressed in BC patients. miR-146a-5p, miR-93-5p, miR-663b, miR-21, miR-4454, miRNA combined diagnostic panel, and urine cytology had certain diagnostic value for BC, with miR-21, miR-4454, and miRNA co-diagnostic panel showing the highest diagnostic value. Collectively, urinary exosomal miR-21 was closely related to Tumor-Node-Metastasis staging and grading in BC patients. Urinary exosomal miR-21 had high diagnostic value for BC and Ta-T1 and T2-T4 stage BC, and had high predictive value for BC poor prognosis, providing an effective indicator for the occurrence, development, and prognostic assessment of BC.

Keywords: bladder cancer; diagnosis; microRNA‐21; prognosis; urinary exosome.

FIGURE 1

Identification of urinary exosomes. (A) Morphological structure of exosomes observed using transmission electron microscopy (TEM); (B) particle size distribution of exosomes analyzed by nanoparticle tracking analysis (NTA); (C) protein expression levels of exosome positive markers CD9, CD63, TSG101, and negative marker Calnexin tested by Western blot.

FIGURE 2

Upregulation of various miRNAs in urinary exosomes of BC patients. (A–E) Relative expression levels of miR‐146a‐5p (A), miR‐93‐5p (B), miR‐663b (C), miR‐21 (D), and miR‐4454 (E) in exosomes assessed by reverse transcription quantitative polymerase chain reaction (RT‐qPCR). N = 116. Non‐normally distributed measurement data represented by quartile (median value [minimum value, maximum value]). Comparisons between groups made using the Mann–Whitney U test. ****p < 0.0001.

FIGURE 3

High diagnostic value of urinary exosomal miR‐21, miR‐4454, and Emdp‐miR for BC. (A–G) Receiver operating characteristic (ROC) curve analysis of miR‐146a‐5p (A), miR‐93‐5p (B), miR‐663b (C), miR‐21 (D), miR‐4454 (E), urine cytology (F), and Emdp‐miR (G) for BC diagnostic value.

FIGURE 4

Diagnostic value of urinary exosomal miR‐21, miR‐4454 and urine cytology for BC at Ta‐T1 and T2‐T4 stage by ROC curve analysis.

FIGURE 5

Association between urinary exosomal miR‐21 level and TNM staging and pathological grading of BC. (A) Relative expression of urinary exosomal miR‐21 in BC patients of different age groups assessed by RT‐qPCR, with 58 years as median age; (B) relative expression of urinary exosomal miR‐21 in male and female BC patients tested by RT‐qPCR; (C) relative expression of urinary exosomal miR‐21 in BC patients at Ta‐T1 stage or T2‐T4 stage determined by RT‐qPCR; (D) relative expression of urinary exosomal miR‐21 in BC patients with PUNLMP/low grade or high grade measured by RT‐qPCR. Measurement data of non‐normal distribution expressed by quartile (median value [minimum, maximum]). Comparisons between groups were performed using the Mann–Whitney U test. NS, no significant difference; **p < 0.001, *p < 0.05.

FIGURE 6

Relationship between miR‐21 and prognosis of patients with BC. Recurrence or death considered as a poor prognosis. ROC curve analysis was used to analyze the predictive value of urinary exosomal miR‐21 for poor prognosis. Using cut‐off value urinary exosomal miR‐21 as a threshold, BC patients divided into low‐miR‐21 and high‐miR‐21 groups, and 3‐year overall survival rates of BC patients in the two groups analyzed by Kaplan–Meier survival curves.