Extracellular vesicle-mediated transmission of circPDLIM5 promotes lymphatic metastasis in prostate cancer

Figures

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  Fig. 1. The characterization and validation of circPDLIM5 in PCa cells. A The cluster heat map showed the 576 differentially expressed circRNAs in 5 pairs of urinary EVs with LN metastasis and without LN metastasis. The red and blue strips indicated up-regulated and down-regulated circRNAs, respectively. B The overlapping analysis of up-regulated circRNAs in the urinary EVs of PCa patients compared with BPH patients and in the LN-positive patients compared with the LN-negative patients. C Schematic illustration revealing circPDLIM5 derived from exons 3 to 8 of PDLIM5. D Rolling circle reverse transcription and Sanger sequencing were used to confirm the full length of circPDLIM5 in PC3 cells. The arrow indicated the special back splicing junction site of circPDLIM5. E Divergent and convergent primers were used to detect circular RNAs in cDNA and gDNA, divergent primers can detect the existence of circular RNAs in cDNA but not in gDNA. GAPDH was used as a control for a linear RNA transcript (F and I). The abundances of circPDLIM5 and PDLIM5 mRNA were determined by PCR and qRT-PCR after treatment with RNase R in PC3 and 22RV1 cells. G and H Cytoplasmic-nuclear fractionation assay and FISH…

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  Fig. 2. The up-regulation of circPDLIM5 is highly associated with PCa lymphatic metastasis. A qRT-PCR analysis of circPDLIM5 expression in PCa patients (n = 327) according to the LN status. B qRT-PCR analysis for the expression level of circPDLIM5 in the metastatic lymph nodes and paired primary PCa tissues (n = 62). C Representative images for the circPDLIM5 expression in LN(+) tissues (n = 62) and LN(-) tissues (n = 265) via FISH. Scale bars: 50 μm. D The percentages of circPDLIM5 expression in PCa tissues (with or without LN metastasis) and normal adjacent tissues (NAT). E Representative images showing circPDLIM5 expression in PCa tissues (with or without LN metastasis) and NAT by in situ hybridization (n = 327). Scale bars: 50 μm. The black rectangle inset is a magnification of the red rectanglearea. F Representative images of ISH and IHC showing the expression of circPDLIM5, LYVE-1 and VEGF-C in PCa tissues (n = 327). The black rectangle inset is a magnification of the red rectanglearea.The Pearson correlation analysis of circPDLIM5 and LYVE-1 (G), circPDLIM5 and VEGF-C (H) displayed that circPDLIM5 was positively correlated with lymphatic vessel density, but no correlation was…

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  Fig. 3. circPDLIM5 is enriched in EVs derived from PCa cells. A The expression level of circPDLIM5 in urinary EVs from 327 PCa patients according to the LN status. B and C TEM and NanoSight were used to identify the character of EVs purified from PC3 cells. Scale bars: 100 nm. D Western blot analysis of EVs-markers from PC3 EVs or cell lysates. E qRT-PCR analysis for the expression level of circPDLIM5 in the EV and non-EV fractions. F qRT-PCR analysis for the expression level of circPDLIM5 in the medium of PCa cells treated with RNase A (2 mg/mL) or in combination with Triton X-100 (0.1%) for 30 min. miR-39-3P was used as an external control. qRT-PCR analysis for the expression level of circPDLIM5 in the CM of PCa cells depleted of EVs by GW4869 (an inhibitor of EVs secretion,20 μM) (G) or by ultracentrifugation (H). miR-39-3P was used as an external control. I and J qRT-PCR analysis of circPDLIM5 expression after knockdown of circPDLIM5 in PCa cells and their corresponding EVs. K and L qRT-PCR analysis of circPDLIM5 expression after up-regulation of circPDLIM5 in PCa cells and their corresponding EVs. Error bars represent the standard deviation (SD) of three independent experiments. **P…

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  Fig. 4. EVs-circPDLIM5 promotes lymphatic metastasis in vivo. A Image of the popliteal LN metastasis model in nude mice. Representative bioluminescence images (B) and mean photon radiance analysis (C) of metastatic popliteal lymph nodes from nude mice treated with PBS, PC3-EVsVector, or PC3-EVscircPDLIM5 after inoculated with PC3 cells into the footpad (n = 10 per group). The white arrow represented the footpad tumor and the red arrow represented the metastatic popliteal lymph node. Representative images of dissected popliteal lymph nodes (D), the volume of lymph nodes (E), and the weight of lymph nodes (F) in all groups. G Percentages of LN metastasis in all groups (n = 10 per group). H Representative images of HE and IHC staining by anti-luciferase antibody in LNs (n = 10 per group). Scale bars: HE 50 μm, IHC 500 μm (up) and 100 μm (down). I Representative images of IHC staining by anti-LYVE-1 anti-body in footpad tumors (n = 10 per group).Scale bars: 50 μm (up) and 20 μm (down). *P < 0.05; **P < 0.01;***P < 0.001. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests (C, E and F), x.2 test (G)

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  Fig. 5. EVs-circPDLIM5 promotes lymphangiogenesis and migration of HLECs in vitro. A-C Representative images of HLECs cultured with PBS, PC3-EVs and 22RV1-EVs. Scale bars: 50 μm. B The length of the formation tubes and (C) the number of Transwell migration cells. D-F Representative images of HLECs cultured with 22RV1-EVssh-NC, 22RV1-EVssh-circPDLIM5#1 and 22RV1-EVssh-circPDLIM5#2. Scale bars: 50 μm. E The length of the formation tubes and (F) the number of Transwell migration cells. G-I Representative images of HLECs cultured with PC3-EVsVector and PC3-EVscircPDLIM5. Scale bars: 50 μm. H The length of the formation tubes and (I) the number of Transwell migration cells. Error bars represent the standard deviation (SD) of three independent experiments. *P < 0.05; **P < 0.01;***P < 0.001. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests (B, C, E and F), 2-tailed Student’s t test (H and I)

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  Fig. 6. The direct interaction between circPDLIM5 and hnRNPA2B1. A Schematic illustration of circPDLIM5 pull-down following mass spectrometry (MS) toidentify proteins interacting with circPDLIM5. B Mass spectrometry analysis revealed that circPDLIM5 could interact with hnRNPA2B1. C qRT-PCR analysis confirmed that circPDLIM5 probe could enrich circPDLIM5 through RNA pull-down assay in PC3 cells. D CircPDLIM5 pull-down assay and Western blot assay in PC3 cells or in PC3-EVs validated the interaction between circPDLIM5 and hnRNPA2B1. E Subcellular co-localization of circPDLIM5 and hnRNPA2B1 in PCa cells measured by fluorescence staining assay. Scale bars: 10 μm. F The enrichment of circPDLIM5 through RIP assay by anti-hnRNPA2B1 antibody in PC3 cells. IgG was used as a negative control. G The binding region of circPDLIM5 to hnRNPA2B1 was predicted by catRAPID. H RIP assay was used to validate the binding site after mutating the 851–927 nt region of circPDLIM5 in PC3 cells. I qRT-PCR analysis for the expression level of circPDLIM5 in EVs when knockdown of hnRNPA2B1 in PCa cells. J qRT-PCR analysis of circPDLIM5 in EVs when mutating the binding sites of circPDLIM5 to hnRNPA2B1 in PCa…

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  Fig. 7. hnRNPA2B1 regulates the package of circPDLIM5 into EVs and EVs-circPDLIM5 can be absorbed by HLECs. A Representative images of HLECs after incubation with EVs derived from PCa cells labeled with PKH67. Scale bars: 10 μm. B The expression level of circPDLIM5 in HLECs when incubated with PBS, PC3-EVs and 22RV1-EVs through qRT-PCR. C The expression level of circPDLIM5 in HLECs when incubated with 22RV1-EVssh-NC, 22RV1-EVssh-circPDLIM5#1 and 22RV1-EVssh-circPDLIM5#2 through qRT-PCR. qRT-PCR analysis of PDLIM5 mRNA expression in HLECs after incubated with 22RV1-EVssh-circPDLIM5 (D) and PC3-EVscircPDLIM5 (E). F–H Representative images for HLECs-circPDLIM5-WT and HLECs-circPDLIM5-KO of tube formation assay and Transwell migration assay. Scale bars: 50 μm. G The length of the formation tubes and (H) the number of Transwell migration cells. I-K Representative images of HLECs (circPDLIM5-WT or circPDLIM5-KO) cultured with 22RV1-EVssh-NC and 22RV1-EVssh-circPDLIM5#2. Scale bars: 50 μm. J The length of the formation tubes and (K) the number of Transwell migration cells. Error bars represent the standard deviation (SD) of three independent experiments. *P < 0.05; **P < 0.01. Statistical…

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  Fig. 8. CircPDLIM5 recruits YY1 to the promoter of PROX1. qRT-PCR analysis (A) and Western blot assay (B) showed the PROX1 expression level after HLECs incubated with PBS, 22RV1-EVsVector and 22RV1-EVscircPDLIM5. C Schematic illustration of circPDLIM5 pull-down assay followed by mass spectrometry analysis in HLECs treated with 22RV1-EVscircPDLIM5 revealed that circPDLIM5 could interact with YY1. D Western blot assay for circPDLIM5 pull-down in HLECs treated with 22RV1-EVscircPDLIM5 confirmed the interaction between circPDLIM5 and YY1. E Subcellular co-localization of circPDLIM5 and YY1 in HLECs incubated with PC3-EVscircPDLIM5 and 22RV1-EVscircPDLIM5 as measured by fluorescence staining assay. Scale bars: 10 μm. F RIP assay through anti-YY1 antibody in HLECs cocultured with 22RV1-EVscircPDLIM5. IgG was used as a negative control. G The binding region of circPDLIM5 to YY1 was predicted by RNAInter. H RIP assay was used to validate the binding site of circPDLIM5 to YY1 after mutating the 695–710 nt region of circPDLIM5 in HLECs treated with 22RV1-EVscircPDLIM5. qRT-PCR analysis (I) and Western blot assay (J) showed the expression level of PROX1 in HLECs cocultured with 22RV1-EVscircPDLIM5…

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  Fig. 9. PROX1 is crucial for the EVs-circPDLIM5 induced lymphangiogenesis. A Representative images of dissected popliteal lymph nodes from nude mice treated with PBS, PC3-EVsVector, and PC3-EVscircPDLIM5 with or without αVEGF-C after inoculated with PC3 cells into the footpad (n = 10 per group). Representative images of the volume of lymph nodes (B), and the weight of lymph nodes (C) in all groups. Representative bioluminescence images (D) and mean photon radiance analysis (E) of metastatic popliteal lymph nodes from nude mice (n = 10 per group). The white arrow represented the footpad tumor and the red arrow represented the metastatic popliteal lymph node. F Percentages of LN metastasis in all groups (n = 10 per group). G Representative images of IHC staining by anti-LYVE-1 anti-body and anti-PROX1 anti-body in footpad tumors (n = 10 per group). Scale bars: 50 μm. The analysis of lymphatic vessel density based on LYVE-1 (H) and the H-score of PROX1 (I) in footpad tumors (n = 10 per group). *P < 0.05; **P < 0.01;***P < 0.001. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests (B, C, E, H and I), x.2 test (F)

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  Fig. 10. EVs-circPDLIM5 is closely related to lymphatic metastasis of PCa. A Representative images of circPDLIM5 expression in PCa tissues by ISH according to the expression level of circPDLIM5 in urinary EVs and serum EVs (n = 327). Scale bars: 50 μm. The black rectangle inset is a magnification of the red rectanglearea. B Correlation analysis of urinary EVs-circPDLIM5 and H-score of circPDLIM5 in tissues (n = 327). C Correlation analysis of serum EVs-circPDLIM5 and H-score of circPDLIM5 in tissues (n = 327). D qRT-PCR analysis of serum EVs-circPDLIM5 in PCa patients (n = 327) according to the LN status. E ROC curve analysis to assess the diagnostic potential of urinary EVs-circPDLIM5 and serum EVs-circPDLIM5 for PCa with LN metastasis. F Kaplan–Meier curves of progression-free survival according to the expression level of urinary EVs-circPDLIM5 in PCa patients (n = 327). G Kaplan–Meier curves of progression-free survival according to the expression level of serum EVs-circPDLIM5 in PCa patients (n = 327). H A schematic model showing the mechanism of PCa cell-derived EVs-circPDLIM5 promoting lymphangiogenesis and LN metastasis by up-regulation of PROX1 in HLECs. ***P < 0.001. Statistical…