circEHBP1 promotes lymphangiogenesis and lymphatic metastasis of bladder cancer via miR-130a-3p/TGFβR1/VEGF-D signaling

Abstract

Lymphatic metastasis constitutes a leading cause of recurrence and mortality in bladder cancer. Accumulating evidence indicates that lymphangiogenesis is indispensable to trigger lymphatic metastasis. However, the specific mechanism is poorly understood. In the present study, we revealed a pathway involved in lymphatic metastasis of bladder cancer, in which a circular RNA (circRNA) facilitated lymphangiogenesis in a vascular endothelial growth factor C (VEGF-C)-independent manner. Novel circRNA circEHBP1 was markedly upregulated in bladder cancer and correlated positively with lymphatic metastasis and poor prognosis of patients with bladder cancer. circEHBP1 upregulated transforming growth factor beta receptor 1 (TGFBR1) expression through physically binding to miR-130a-3p and antagonizing the suppression effect of miR-130a-3p on the 3' UTR region of TGFBR1. Subsequently, circEHBP1-mediated TGFβR1 overexpression activated the TGF-β/SMAD3 signaling pathway, thereby promoting the secretion of VEGF-D and driving lymphangiogenesis and lymphatic metastasis in bladder cancer. Importantly, administration of VEGF-D neutralizing antibodies remarkably blocked circEHBP1-induced lymphangiogenesis and lymphatic metastasis in vivo. Our findings highlighted that the circEHBP1/miR-130a-3p/TGFβR1/VEGF-D axis contributes to lymphangiogenesis and lymphatic metastasis of bladder cancer independent of VEGF-C, which might lead to the development of circEHBP1 as a potential biomarker and promising therapeutic target for lymphatic metastasis in bladder cancer.

Keywords: TGF-β pathway; VEGF-D; bladder cancer; circRNA; lymphangiogenesis.

Graphical abstract

Figure 1

circEHBP1 is upregulated in LN metastasis of BCa (A) Schematic illustration of the modeling of poorly invasive and highly invasive cell sublines from BCa. (B) Relative expression of circEHBP1 in poorly invasive, wild-type, and highly invasive UM-UC-3 and T24 cell sublines. (C) circEHBP1 level analyzed by quantitative real-time RT-PCR in a 17-case cohort of LN metastatic BCa tissues with low VEGF-C expression paired with corresponding NATs. The Mann-Whitney U test was employed. (D) Relative expression of circEHBP1 in BCa cell lines and SV-HUC-1 was detected using quantitative real-time RT-PCR. (E) Quantitative real-time RT-PCR for the circEHBP1 expression in a 186-case cohort of BCa tissues paired with corresponding NATs. The Mann-Whitney U test was employed. (F and G) Quantitative real-time RT-PCR analysis of circEHBP1 expression in 186 BCa tissues with respect to LN status (F) and pathological grade (G). The Mann-Whitney U test was employed. (H) circEHBP1 expression in primary BCa samples and paired metastatic LNs was detected by quantitative real-time RT-PCR. The Mann-Whitney U test was employed. (I and J) Kaplan-Meier survival curves for OS (I) and DFS (J) of low circEHBP1 level versus high circEHBP1 level in patients with BCa. The median expression level of circEHBP1 was taken as the cutoff value. (K and L) Representative images (K) and proportion (L) for IHC staining showing the LVD stained with anti-LYVE-1 in the BCa tissues with differential circEHBP1 expression. Scale bar, 50 μm. (M) Schematic illustrating the genetic locus of the EHBP1 gene and the circEHBP1 derived from exon 15 to 19 of EHBP1. (N) Sanger sequencing for the back-splice junction site of circEHBP1. (O and P) PCR with an agarose gel electrophoresis assay for circEHBP1 and EHBP1 in the cDNA and gDNA of UM-UC-3 (O) or T24 (P) cells. GAPDH was applied for NC. (Q) Relative circEHBP1 expression was evaluated by quantitative real-time RT-PCR using random primers or oligo-dT primers. (R) circEHBP1 and EHBP1 mRNA expression analyzed by quantitative real-time RT-PCR following RNase R treatment in UM-UC-3 and T24 cells. (S and T) Actinomycin D assay to assess the stability of circEHBP1 and EHBP1 mRNA in UM-UC-3 (S) or T24 (T) cells at the indicated time points. Two-tailed Student’s t test or 1-way analyses of variance were used for significance level, and Dunnett’s test was used to perform multiple comparisons. Error bars indicate the standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 2

circEHBP1 promotes lymphangiogenesis of BCa in vitro (A–D) circEHBP1 and EHBP1 level were assessed using quantitative real-time RT-PCR in circEHBP1 knockdown (A and B), circEHBP1 overexpression (C and D), and paired control BCa cells. (E–J) Representative images (E and H) and quantification of tube formation (F and I) and Transwell migration (G and J) by HLECs that were cultured with the supernatant obtained from control or circEHBP1-silencing UM-UC-3 and T24 cells. Scale bars, 100 μm. (K–P) Representative images (K and N) and quantification of tube formation (L and O) and Transwell migration (M and P) by HLECs were cultured with the supernatant obtained from control or circEHBP1-overexpressing UM-UC-3 and T24 cells. Scale bars, 100 μm. Two-tailed Student’s t test or 1-way analyses of variance were used to determine the significance level, and Dunnett’s test was used for multiple comparisons. Error bars indicate the standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 3

circEHBP1 facilitate LN metastasis of BCa in vivo (A) Representative images of popliteal LN metastasis in the nude mouse model. UM-UC-3 cells were inoculated into the right footpads of the nude mice, followed by the enucleation and evaluation of popliteal LNs. (B and C) Representative bioluminescence images (B) and histogram analysis (C) of popliteal LN metastasis from nude mice (n = 12) following overexpressing circEHBP1. (D and E) Representative images (D) of excised popliteal LNs from nude mice (n = 12) and the measurement of the LN volume (E). (F) The popliteal LN metastatic rate in all groups (n = 12). (G and H) Representative images (G) and histogram analysis (H) of IHC staining showing the LVD stained with anti-LYVE-1 in the mouse tissues with different circEHBP1 expression (n = 12). Scale bar, 50 μm. (I) Kaplan-Meier survival curves in all groups (n = 12). Two-tailed Student’s t test or 1-way analyses of variance were used to determine the significance level and Dunnett’s test for multiple comparisons. Error bars indicate standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 4

circEHBP1 functions as a miR-130-3p sponge in BCa (A) The cellular localization of circEHBP1 was detected using a FISH assay. Scale bar, 100 μm. (B) The cellular localization of circEHBP1 in UM-UC-3 cells was confirmed using a subcellular fractionation assay. The nuclear control used U6 and the cytoplasmic control used 18S rRNA. (C) RegRNA 2.0 was used to predict the potential target miRNAs of circEHBP1. (D and E) Quantitative real-time RT-PCR analysis for the expression of ten predicted target miRNAs of circEHBP1 in UM-UC-3 (D) and T24 (E) cells. (F) RNAalifold was used to predict the secondary structure of circEHBP1. (G) Schematic illustrating the sequence alignment of circEHBP1 with miR-130a-3p. (H) The luciferase activities of the circEHBP1-wt plasmid or circEHBP1-mut plasmid quantified following transfecting NC mimic or miR-130a-3p mimics into UM-UC-3 cells. (I) Quantitative real-time RT-PCR analysis of the circEHBP1 captured by miR-130a-3p. (J) The co-localization of circEHBP1 and miR-130a-3p was detected by FISH assay. Scale bar, 100 μm. Two-tailed Student’s t test or 1-way analyses of variance were used to determine the significance level, and Dunnett’s test was used for multiple comparisons. Error bars indicate standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 5

circEHBP1 attenuates miR-130a-3p-mediated TGFBR1 suppression (A–D) Representative images and quantification of tube formation and Transwell migration by HLECs that were cultured with the supernatant obtained from miR-130a-3p-silencing (A and B), miR-130a-3p-overexpressing (C and D), or control UM-UC-3 and T24 cells. Scale bars, 100 μm. (E) Schematic illustrating the sequence alignment of miR-130a-3p with the 3′ UTR of TGFβR1. (F and G) The luciferase activities of the TGFβR1-3′ UTR-wt plasmid or TGFβR1-3′ UTR-mut plasmid quantified following transfection with the NC mimic or miR-130a-3p mimics in UM-UC-3 (F) and T24 (G) cells. (H and I) Quantitative real-time RT-PCR analysis of the effect of circEHBP1 knockdown (H) or circEHBP1 overexpression (I) on TGFBR1 expression in UM-UC-3 and T24 cells. (J) The effect of circEHBP1 knockdown on miR-130a-3p depletion-induced TGFβR1 promotion in UM-UC-3 and T24 cells was analyzed by quantitative real-time RT-PCR. (K and L) Western blotting analysis of the effect of circEHBP1 knockdown on miR-130a-3p depletion-induced TGFβR1 expression in UM-UC-3 (K) and T24 (L) cells. (M–P) Western blotting assay showing the levels of TGFβR1, SMAD3, and p-SMAD3 after circEHBP1 knockdown (M and N) or circEHBP1 overexpression (O and P) in UM-UC-3 and T24 cells. Two-tailed Student’s t test or 1-way analyses of variance were used to determine the significance level, and Dunnett’s test was used for multiple comparisons. Error bars indicate the standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 6

circEHBP1 elevates VEGF-D expression via activating the TGF-β/SMAD3 signaling pathway (A and B) Quantitative real-time RT-PCR analysis of the impact of circEHBP1 knockdown (A) or circEHBP1 overexpression (B) on the VEGFD expression in UM-UC-3 and T24 cells. (C and D) ELISA analysis of the impact of circEHBP1 knockdown (C) or circEHBP1 overexpression (D) on VEGF-D secretion in UM-UC-3 and T24 cells. (E and F) Quantitative real-time RT-PCR analysis of the treatment effect of LY364947 on circEHBP1-overexpressing-induced VEGFD expression in UM-UC-3 (E) and T24 (F) cells. (G and H) ELISA analysis of the treatment effect of LY364947 on circEHBP1-overexpressing-induced VEGF-D secretion in UM-UC-3 (G) and T24 (H) cells. (I) Representative images and quantification of tube formation and Transwell migration by HLECs that were cultured with the supernatant obtained from UM-UC-3 cell with vector, circEHBP1-overexpressing, circEHBP1-overexpressing+PBS, and circEHBP1-overexpressing +αVEGF-D. Scale bar, 100 μm. (J) The measurement of the LN volume in the indicted groups. (K) The popliteal LN metastatic rate in all groups (n = 12). (L) Kaplan-Meier survival curves in all groups (n = 12). (M–O). Representative images (M) and histogram analysis (N and O) of IHC staining showing the effects of VEGF-D neuralization on circEHBP1-induced VEGF-D expression and the quantities of lymphatic vessels in footpad tumors. Scale bar, 50 μm. Two-tailed Student’s t test or 1-way analyses of variance were used to determine the significance level, and Dunnett’s test was used for multiple comparisons. Error bars indicate standard deviations. ∗p < 0.05, ∗∗p < 0.01.

Figure 7

Clinical significance of circEHBP1 inducing miR-130a-3p/TGFBR1/VEGF-D axis in patients with BCa (A) Quantitative real-time RT-PCR for the miR-130a-3p level among a 186-case cohort of BCa tissues paired with corresponding NATs. The Mann-Whitney U test was employed. (B and C) Quantitative real-time RT-PCR analysis of miR-130a-3p levels among 186 cases of BCa with respect to LN status (B) and pathological grade (C). The Mann-Whitney U test was employed. (D) Quantitative real-time RT-PCR for the VEGF-D expression in a 186-case cohort of BCa tissues paired with corresponding NATs. The Mann-Whitney U test was employed. (E) Quantitative real-time RT-PCR analysis of VEGF-D expression in 186 cases of BCa according to LN status. The Mann-Whitney U test was employed. (F and G) Kaplan-Meier survival curves for OS (F) and DFS (G) of low miR-130a-3p versus high miR-130a-3p level in patients with BCa. The median expression of miR-130a-3p was taken as the cutoff value. (H) Quantitative real-time RT-PCR for the correlation of circEHBP1 and VEGF-D expression in 186 cases of BCa. (I) Quantitative real-time RT-PCR for the correlation of miR-130a-3p and VEGF-D expression in 186 cases of BCa. (J) Schematic illustrating the potential mechanism of circEHBP1 on lymphangiogenesis and LN metastasis facilitation in BCa via the miR-130a-3p/TGFβR1/VEGF-D axis. Error bars indicate standard deviations. ∗p < 0.05, ∗∗p < 0.01.