LncRNA BCYRN1 as a Potential Therapeutic Target and Diagnostic Marker in Serum Exosomes in Bladder Cancer

Abstract

Bladder cancer (BC) is a common genitourinary malignancy that exhibits silent morbidity and high mortality rates because of a lack of diagnostic markers and limited effective treatments. Here, we evaluated the role of the lncRNA brain cytoplasmic RNA 1 (BCYRN1) in BC. We performed loss-of-function assays to examine the effects of BCYRN1 downregulation in T24 and BOY BC cells. We found that BCYRN1 downregulation significantly inhibited the proliferation, migration, invasion, and three-dimensional spheroid formation ability and induced apoptosis in BC cells. Additionally, gene set enrichment analysis (GSEA) using RNA sequences from tumor fractions showed that BCYRN1 downregulation decreased the expression of mRNAs associated with the cell cycle. These findings were supported by observations of G₂/M arrest in flow cytometry assays. Finally, we examined the expression of serum exosomal BCYRN1 as a biomarker. Clinically, BCYRN1 expression in serum exosomes from patients with BC (n = 31) was significantly higher than that in healthy donors (n = 19; mean difference: 4.1-fold higher, p < 0.01). Moreover, in patients who had undergone complete resection of BC, serum exosomal BCYRN1 levels were significantly decreased (n = 8). Thus, serum exosomal BCYRN1 may be a promising diagnostic marker and therapeutic target in patients with BC.

Keywords: BCYRN1; biomarker; bladder cancer; exosome; lncRNA.

Figure 1

Knockdown of BCYRN1 decreased the viability of BC cells. (A) Cell proliferation in two-dimensional monolayer culture was evaluated using XTT assays in BC cell lines. (B) Cell proliferation in 3D cell culture was evaluated using 3D spheroid assays in BC cell lines. The proliferation of BC cells was significantly reduced by the knockdown of BCYRN1. (C) Cell migration ability was measured using wound healing assays. Migration ability was significantly reduced by the knockdown of BCYRN1. (D) Cell invasion ability was measured using Matrigel invasion assays. The counted infiltrated cells were decreased in BCYRN1-knockdown cells compared with those in mock-transfected cells. Each experiment was repeated at least three times. The error bars indicate standard errors of the means. * p < 0.0001. The relationships among the four groups were analyzed using multiple comparison tests with the Bonferroni–Dunn method.

Figure 2

BCYRN1 knockdown decreased BC cell viability in a xenograft mouse model. (A) Comparison of tumor volumes in mice subcutaneously inoculated with siNC- or siBCYRN1-transfected T24 cells (n = 5, p = 0.0139, Mann–Whitney U test). (B) Photograph showing excised tumor tissue from the xenograft mouse model on day 31. The graph on the right side shows significant differences between the groups (p = 0.038, Mann–Whitney U test). (C) Photograph showing Ki-67-positive cells in the excised tumor stained using immunohistochemistry (200×). The graph on the right side indicates the percentage of Ki-67-positive cells (p < 0.0001, Mann–Whitney U test). The error bars indicate standard errors of the means.

Figure 3

BCYRN1 downregulation affected the cell cycle and cell apoptosis. (A) Hallmark gene enrichment analysis of downregulated gene expression events in T24 and BOY cells transfected with siBCYRN1-1. The color shows the p-value, the y-axis shows the NES, and the circle size indicates the gene ratio. The lower panel shows each of the enriched gene set events. (B,C) Western blot analysis of cleaved PARP, CDK2, and Bcl-xL expression in cells transfected with siBCYRN1 or siNC.

Figure 4

Knockdown of BCYRN1 induced G₂/M arrest and apoptosis in BC cells. (A) The percentage of cells in each cell cycle phase among groups of cells transfected with siBCYRN1 or siNC (* p < 0.005). (B) The percentage of apoptotic cells, as determined using flow cytometry, among cells transfected with siBCYRN1 or siNC (* p < 0.001). The relationships among the four groups were analyzed using multiple comparison tests with the Bonferroni–Dunn method.

Figure 5

High serum exosomal BCYRN1 levels were observed with the presence of BC tumors. (A) Method for the isolation of exosomes from patient serum using ultracentrifugation. (B) Comparison of serum exosomal BCYRN1 expression levels between patients with BC and healthy donors (p = 0.0085, Mann–Whitney U test). The error bars indicate standard errors of the means. The horizontal bar indicates the mean of the samples. (C) Comparison of serum exosomal BCYRN1 expression levels before and after TURBT in the same patient. The solid line represents patients without residual tumors, in whom expression of BCYRN1 significantly decreased after completion, and the dashed line represents the patients with residual tumors on the second TUR. (D) Schema of this study.