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Exosomal circSPIRE1 mediates glycosylation of E-cadherin to suppress metastasis of renal cell carcinoma

Shu, Guannan, Lu, Xuanxuan, Pan, Yihui, Cen, Junjie, Huang, Kangbo, Zhou, Mi, Lu, Jun, Dong, Jiaqi, Han, Hui, Chen, Wei, Lin, Juan, Luo, Junhang, Zhang, Jiaxing·2023·Oncogene
DOI 10.1038/s41388-023-02678-7PMC PMC10238271
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Abstract

Metastasis is the main cause of mortality in renal cell carcinoma (RCC). Circular RNAs (circRNAs) involvement in RCC metastasis has been described, although the underlying mechanisms remain unknown. We evaluated recurring lung-metastasis cases using patient-derived xenograft models and isolated a highly metastatic clone. CircSPIRE1 was identified as a metastasis-inhibiting circRNA in clinical cohort and xenograft models. Mechanistically, circSPIRE1 suppressed mesenchymal state through regulating ELAV like RNA binding protein 1-mRNA binding, and upregulating polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) and KH domain RNA binding protein (QKI) expression. GALNT3 promoted glycosylation and cytomembrane localization of E-cadherin. QKI formed a positive feedback loop to enhance circSPIRE1 expression. Meanwhile, exosomal circSPIRE1 suppressed angiogenesis and vessel permeability. Our work reveals a non-canonical route for circRNAs in RCC to suppress metastasis. Furthermore, a nanomedicine consisting of circSPIRE1 plasmid suppressed metastasis formation. In conclusion, circSPIRE1 may be a predictor of metastasis and a potential therapeutic target of metastatic RCC.

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Figures

Fig. 1 Downregulation and characterization of circSPIRE1 in metastatic RCC. A Schematic illustration of the development of the metastatic RCC PDX model, PDX LM. B Increased tumor metastasis formed in lungs of PDX LM model compared to the original PDX model. Left, representative lung and representative HE staining of lung metastatic lesions, scale bar, 100 µm. Right, the number of metastatic nodules formed in the lungs of mice for each group (n = 6 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. C Increased microvessel density of the PDX LM model compared to the original PDX model. Left, representative tumor stained with CD34 anti-body. Right, microvessel density for each group (n = 5 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. D Heatmap of high-throughput sequencing between PDX LM cells and original PDX cells. E Volcano plot of circRNAs comparing five no clinically metastasis cases and five metastasis cases. F qRT-PCR analysis of four circRNAs expression in 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. G Left, flowchart illustrating the screening criteria of potential metastasis related circRNAs. Right, Kaplan−Meier analysis of DFS in RCC patients with low versus high expression of circSPIRE1. The P value was determined by a Log-rank test. H Existence of circSPIRE1 confirmed by RT-PCR and gel electrophoresis using convergent and divergent primers. CircSPIRE1 can only be amplified in cDNA. I Northern blotting of circSPIRE1 and SPIRE1 transcripts by hybridization with exon 4 (left) and exon 6−exon 4 junction (right) probes in the presence or absence of RNase R treatment. J RNA fluorescence in situ hybridization for circSPIRE1. Scale bar, 10 µm. K Cytoplasmic and nuclear RNA fractionation experiment showing that circSPIRE1 mainly localized in the cytoplasm. GAPDH and U3 as positive controls in the cytoplasm and nucleus, respectively.
Fig. 1 Downregulation and characterization of circSPIRE1 in metastatic RCC. A Schematic illustration of the development of the metastatic RCC PDX model, PDX LM. B Increased tumor metastasis formed in lungs of PDX LM model compared to the original PDX model. Left, representative lung and representative HE staining of lung metastatic lesions, scale bar, 100 µm. Right, the number of metastatic nodules formed in the lungs of mice for each group (n = 6 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. C Increased microvessel density of the PDX LM model compared to the original PDX model. Left, representative tumor stained with CD34 anti-body. Right, microvessel density for each group (n = 5 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. D Heatmap of high-throughput sequencing between PDX LM cells and original PDX cells. E Volcano plot of circRNAs comparing five no clinically metastasis cases and five metastasis cases. F qRT-PCR analysis of four circRNAs expression in 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. G Left, flowchart illustrating the screening criteria of potential metastasis related circRNAs. Right, Kaplan−Meier analysis of DFS in RCC patients with low versus high expression of circSPIRE1. The P value was determined by a Log-rank test. H Existence of circSPIRE1 confirmed by RT-PCR and gel electrophoresis using convergent and divergent primers. CircSPIRE1 can only be amplified in cDNA. I Northern blotting of circSPIRE1 and SPIRE1 transcripts by hybridization with exon 4 (left) and exon 6−exon 4 junction (right) probes in the presence or absence of RNase R treatment. J RNA fluorescence in situ hybridization for circSPIRE1. Scale bar, 10 µm. K Cytoplasmic and nuclear RNA fractionation experiment showing that circSPIRE1 mainly localized in the cytoplasm. GAPDH and U3 as positive controls in the cytoplasm and nucleus, respectively.CC BY · PubMed Central
Fig. 2 Function assays of circSPIRE1 in RCC cells. A Transwell assays showing that expression of circSPIRE1 affected the migration ability of RCC cells. Left, transwell representative images. Scale bar, 100 µm. Right, numbers of migration cell (n = 5/group). B Bioluminescent in the lungs of mice through sub-capsular injection showing that expression of circSPIRE1 affected RCC metastasis in vivo. Top, representative bioluminescent images of lungs for each experimental group. Bottom, statistical analysis of bioluminescent tracking plots (n = 6 mice/group). C Tumor metastasis formed in the lungs of mice through sub-capsular injection showing that expression of circSPIRE1 affected RCC metastasis in vivo. Top, representative lung and representative HE staining of lung metastatic lesions. Scale bar, 100 µm. Bottom, statistical analysis of the number of metastatic nodules formed in the lungs of mice for each group (n = 6 mice/group). B, C Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test.
Fig. 2 Function assays of circSPIRE1 in RCC cells. A Transwell assays showing that expression of circSPIRE1 affected the migration ability of RCC cells. Left, transwell representative images. Scale bar, 100 µm. Right, numbers of migration cell (n = 5/group). B Bioluminescent in the lungs of mice through sub-capsular injection showing that expression of circSPIRE1 affected RCC metastasis in vivo. Top, representative bioluminescent images of lungs for each experimental group. Bottom, statistical analysis of bioluminescent tracking plots (n = 6 mice/group). C Tumor metastasis formed in the lungs of mice through sub-capsular injection showing that expression of circSPIRE1 affected RCC metastasis in vivo. Top, representative lung and representative HE staining of lung metastatic lesions. Scale bar, 100 µm. Bottom, statistical analysis of the number of metastatic nodules formed in the lungs of mice for each group (n = 6 mice/group). B, C Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test.CC BY · PubMed Central
Fig. 3 5ʹ-AGAGCGAUGAAUCUAGCACA-3ʹ sequence of circSPIRE1 interacts with RRM3 domain of ELAVL1. A Top, identification of the circSPIRE1-protein complex pulled down by circSPIRE1 junction probe with protein extracts from original PDX cells. Bottom, immunoblot analysis of ELAVL1 after pulldown assay showing its specific association with circSPIRE1. B RIP assays showing the association of ELAVL1 with circSPIRE1. Relative enrichment representing RNA levels associated with ELAVL1 compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. C IF-FISH assay showing that circSPIRE1 is colocalized with ELAVL1 protein in the cytoplasm. Scale bar, 10 µm. D Western blotting assay showing the level of ELAVL1 protein pulled down by biotin-labeled circSPIRE1 probes from the lysates of original PDX cells following transfection with shRNAs targeted circSPIRE1 and negative control. E Relative enrichment representing circSPIRE1 levels associated with truncated ELAVL1 relative to an input control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. F Flowchart illustrating the computational analysis of circSPIRE1 interaction with ELAVL1. G Graphical representation of the best scored three-dimensional structures of circSPIRE1 and ELAVL1 (RRM3 domain) docking models with a zoom-in image. H Refinement of the best docked circSPIRE1-ELAVL1 (RRM3 domain) model showing MC score vs steps of simulation. I RNA-EMSA assay showing the binding ability of purified ELAVL1 with two biotin-labeled oligonucleotides containing two predicted sequence from circSPIRE1. J Western blotting assay showing the level of ELAVL1 protein pulled down by biotin-labeled circSPIRE1 probes from the lysates of 293 T following transfection with circSPIRE1 full-length plasmid or truncated circSPIRE1 plasmid.
Fig. 3 5ʹ-AGAGCGAUGAAUCUAGCACA-3ʹ sequence of circSPIRE1 interacts with RRM3 domain of ELAVL1. A Top, identification of the circSPIRE1-protein complex pulled down by circSPIRE1 junction probe with protein extracts from original PDX cells. Bottom, immunoblot analysis of ELAVL1 after pulldown assay showing its specific association with circSPIRE1. B RIP assays showing the association of ELAVL1 with circSPIRE1. Relative enrichment representing RNA levels associated with ELAVL1 compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. C IF-FISH assay showing that circSPIRE1 is colocalized with ELAVL1 protein in the cytoplasm. Scale bar, 10 µm. D Western blotting assay showing the level of ELAVL1 protein pulled down by biotin-labeled circSPIRE1 probes from the lysates of original PDX cells following transfection with shRNAs targeted circSPIRE1 and negative control. E Relative enrichment representing circSPIRE1 levels associated with truncated ELAVL1 relative to an input control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. F Flowchart illustrating the computational analysis of circSPIRE1 interaction with ELAVL1. G Graphical representation of the best scored three-dimensional structures of circSPIRE1 and ELAVL1 (RRM3 domain) docking models with a zoom-in image. H Refinement of the best docked circSPIRE1-ELAVL1 (RRM3 domain) model showing MC score vs steps of simulation. I RNA-EMSA assay showing the binding ability of purified ELAVL1 with two biotin-labeled oligonucleotides containing two predicted sequence from circSPIRE1. J Western blotting assay showing the level of ELAVL1 protein pulled down by biotin-labeled circSPIRE1 probes from the lysates of 293 T following transfection with circSPIRE1 full-length plasmid or truncated circSPIRE1 plasmid.CC BY · PubMed Central
Fig. 4 CircSPIRE1/ELAVL1/mRNA ternary complex stabilizes GALNT3 and QKI mRNA. A Western blotting (left) as well as qPCR (right) validation of GALNT3 and QKI upon circSPIRE1 knockdown and overexpression. B Relative enrichment representing GALNT3 mRNA (left) and QKI mRNA (right) levels associated with circSPIRE1 junction compared to controls. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. C Reduced stability of GALNT3 (left) and QKI (right) upon circSPIRE1 knockdown. D Relative enrichment representing the enrichment of GALNT3 mRNA (left) and QKI mRNA (right) associated with the truncated ELAVL1 protein complex compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. E IF-FISH assay showing that the colocalization of circSPIRE1/ELAVL1/mRNA was decreased upon knockdown of circSPIRE1. Scale bar, 10 µm. F Relative luciferase activity of luciferase reporter gene with GALNT3-WT or GALNT3-Mut (left) and QKI-WT or QKI-Mut (right). Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P value was determined by Student’s t test. G Upon knockdown or overexpression of circSPIRE1, relative luciferase activity of luciferase reporter gene with GALNT3-WT or GALNT3-Mut (left) and QKI-WT or QKI-Mut (right). Data represent mean ± S.D.; dot plots reflect data points from three independent experiment. The P value was determined by Student’s t test. H Upon knockdown or overexpression of circSPIRE1, relative enrichment representing GALNT3 mRNA (left) and QKI mRNA (right) levels associated with ELAVL1 protein complex compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments.
Fig. 4 CircSPIRE1/ELAVL1/mRNA ternary complex stabilizes GALNT3 and QKI mRNA. A Western blotting (left) as well as qPCR (right) validation of GALNT3 and QKI upon circSPIRE1 knockdown and overexpression. B Relative enrichment representing GALNT3 mRNA (left) and QKI mRNA (right) levels associated with circSPIRE1 junction compared to controls. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. C Reduced stability of GALNT3 (left) and QKI (right) upon circSPIRE1 knockdown. D Relative enrichment representing the enrichment of GALNT3 mRNA (left) and QKI mRNA (right) associated with the truncated ELAVL1 protein complex compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. E IF-FISH assay showing that the colocalization of circSPIRE1/ELAVL1/mRNA was decreased upon knockdown of circSPIRE1. Scale bar, 10 µm. F Relative luciferase activity of luciferase reporter gene with GALNT3-WT or GALNT3-Mut (left) and QKI-WT or QKI-Mut (right). Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P value was determined by Student’s t test. G Upon knockdown or overexpression of circSPIRE1, relative luciferase activity of luciferase reporter gene with GALNT3-WT or GALNT3-Mut (left) and QKI-WT or QKI-Mut (right). Data represent mean ± S.D.; dot plots reflect data points from three independent experiment. The P value was determined by Student’s t test. H Upon knockdown or overexpression of circSPIRE1, relative enrichment representing GALNT3 mRNA (left) and QKI mRNA (right) levels associated with ELAVL1 protein complex compared to an input control. IgG antibody served as a control. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments.CC BY · PubMed Central
Fig. 5 CircSPIRE1 suppresses EMT of RCC cells through stabilizing GALNT3 mRNA. A Kaplan−Meier analysis of DFS in RCC patients with low versus high expression of GALNT3. The P-value was determined by the Log-rank test. B Positive correlation between GALNT3 expression and circSPIRE1 expression in 142 clinical samples of RCC. C qRT-PCR analysis of GALNT3 expression in 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; the P values were determined by Student’s t test. D Transwell assays showing that circSPIRE1 suppresses the migration ability of RCC cells depending on GALNT3. Top, representative images of the Transwell assay. Scale bar, 100 µm. Bottom, numbers of migrating cells (n = 5/group). E Western blots of epithelial and mesenchymal markers upon overexpression of circSPIRE1 in PDX LM and knockdown of circSPIRE1 in original PDX. F Western blots of enhanced epithelial markers upon overexpression of circSPIRE1 was reversed by GALNT3 knockdown in PDX LM and reduced epithelial markers upon knockdown of circSPIRE1 was reversed by GALNT3 overexpression in original PDX. G E-cadherin O-GalNAc glycosylation decreases in PDX LM comparing with original PDX and increases upon circSPIRE1 overexpression. Knockdown of GALNT3 reversed the effect of circSPIRE1 overexpression. H E-cadherin retention in the Golgi increases in PDX LM compared to original PDX and decreases upon circSPIRE1 overexpression. Knockdown of GALNT3 reversed the effect of circSPIRE1 overexpression. Scale bar, 10 µm. Pearson’s coeff was used as quantifications.
Fig. 5 CircSPIRE1 suppresses EMT of RCC cells through stabilizing GALNT3 mRNA. A Kaplan−Meier analysis of DFS in RCC patients with low versus high expression of GALNT3. The P-value was determined by the Log-rank test. B Positive correlation between GALNT3 expression and circSPIRE1 expression in 142 clinical samples of RCC. C qRT-PCR analysis of GALNT3 expression in 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; the P values were determined by Student’s t test. D Transwell assays showing that circSPIRE1 suppresses the migration ability of RCC cells depending on GALNT3. Top, representative images of the Transwell assay. Scale bar, 100 µm. Bottom, numbers of migrating cells (n = 5/group). E Western blots of epithelial and mesenchymal markers upon overexpression of circSPIRE1 in PDX LM and knockdown of circSPIRE1 in original PDX. F Western blots of enhanced epithelial markers upon overexpression of circSPIRE1 was reversed by GALNT3 knockdown in PDX LM and reduced epithelial markers upon knockdown of circSPIRE1 was reversed by GALNT3 overexpression in original PDX. G E-cadherin O-GalNAc glycosylation decreases in PDX LM comparing with original PDX and increases upon circSPIRE1 overexpression. Knockdown of GALNT3 reversed the effect of circSPIRE1 overexpression. H E-cadherin retention in the Golgi increases in PDX LM compared to original PDX and decreases upon circSPIRE1 overexpression. Knockdown of GALNT3 reversed the effect of circSPIRE1 overexpression. Scale bar, 10 µm. Pearson’s coeff was used as quantifications.CC BY · PubMed Central
Fig. 6 CircSPIRE1 expression was dependent on the QKI positive feedback loop. A QKI was associated with the introns flanking circSPIRE1. RIP assays were conducted with a QKI antibody or control IgG, and primer sets covering the introns flanking circSPIRE1 (top). RNA enrichment indicates the levels of the indicated fragments in the anti-QKI precipitates relative to those in the IgG precipitates (bottom). Mean ± S.D. are provided. Dot plots reflect data points from three independent experiments. B Schematic structures showing RNA-binding sequence in the introns flanking the circSPIRE1 and a summary of intron truncations. C RNA pull-down assay performed to analyze the interaction between QKI and introns flanking circSPIRE1. D CircSPIRE1 expression upon QKI knockdown or overexpression. E Positive correlation between QKI expression and circSPIRE1 expression in 142 clinical samples of RCC. F Schematic diagram of mechanism of circSPIRE1 during RCC metastasis in RCC cells.
Fig. 6 CircSPIRE1 expression was dependent on the QKI positive feedback loop. A QKI was associated with the introns flanking circSPIRE1. RIP assays were conducted with a QKI antibody or control IgG, and primer sets covering the introns flanking circSPIRE1 (top). RNA enrichment indicates the levels of the indicated fragments in the anti-QKI precipitates relative to those in the IgG precipitates (bottom). Mean ± S.D. are provided. Dot plots reflect data points from three independent experiments. B Schematic structures showing RNA-binding sequence in the introns flanking the circSPIRE1 and a summary of intron truncations. C RNA pull-down assay performed to analyze the interaction between QKI and introns flanking circSPIRE1. D CircSPIRE1 expression upon QKI knockdown or overexpression. E Positive correlation between QKI expression and circSPIRE1 expression in 142 clinical samples of RCC. F Schematic diagram of mechanism of circSPIRE1 during RCC metastasis in RCC cells.CC BY · PubMed Central
Fig. 7 RCC-secreted circSPIRE1 is transferred to endothelial cells and suppressed vascular permeability and angiogenesis. A Left, qRT-PCR analysis of circSPIRE1 expression in sera of 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. Right, correlation of circSPIRE1 expression between in serum and in primary tumor. B Correlation analysis of circSPIRE1 (red) expression in RCC epithelium and stroma (labeled by CK, purple) and their adjacent endothelial cells (labeled by CD34, green). CircSPIRE1 levels were determined by the FISH score, Spearman rank correlation coefficient (rs), and P value. P value is from Spearman’s test. Scale bar, 50 µm. C Cytoplasmic and exosome RNA fractionation experiment showing that circSPIRE1 mainly localized in the exosome. D Transmission electron microscopy of exosomes derived from original PDX and PDX LM. E Exosome marker TSG101 and CD63 analyzed through western blotting. F RT-PCR analysis of circSPIRE1 expression in HUVECs incubated with exosomes derived from p-CTRL and p-circSPIRE1 cells for 3, 6, 12, 24, and 48 h. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. G Effect of p-CTRL, p-circSPIRE1 and p-circSPIRE1 + Annexin V treatments on circSPIRE1 expression in HUVECs. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. H Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on tube formation ability of HUVECs by tube formation assay. Mean ± S.D. are provided (n = 3/group). Scale bar represents 100 µm. I Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on vascular outgrowth of rat aortic rings. Vascular outgrowth was quantified by counting all sprouts from one ring. Left, representative aortic ring images (n = 3/group). Data represent mean ± S.D.; dot plot reflects data points from three independent experiments. Scale bar represents 200 µm. J Permeability of the HUVEC monolayers to rhodamine–dextran (70 kDa) after exposure to exosomes derived from circSPIRE1 knockdown or overexpression cells for 72 h. Mean ± S.D. are shown. Dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. K Effect of circSPIRE1 knockdown or overexpression on tumor microvessel genesis. Top, representative tumor stained with CD34 anti-body. Bottom, microvessel density for each group. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. The P values were determined by Student’s t test. L VEGFR2, Occluding and Claudin5 protein expression in circSPIRE1 overexpression or circSPIRE1 overexpression combined GALNT3 knockdown HUVECs by western blot. M Schematic diagram of mechanism of circSPIRE1 during RCC metastasis in tumor microenvironment.
Fig. 7 RCC-secreted circSPIRE1 is transferred to endothelial cells and suppressed vascular permeability and angiogenesis. A Left, qRT-PCR analysis of circSPIRE1 expression in sera of 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. Right, correlation of circSPIRE1 expression between in serum and in primary tumor. B Correlation analysis of circSPIRE1 (red) expression in RCC epithelium and stroma (labeled by CK, purple) and their adjacent endothelial cells (labeled by CD34, green). CircSPIRE1 levels were determined by the FISH score, Spearman rank correlation coefficient (rs), and P value. P value is from Spearman’s test. Scale bar, 50 µm. C Cytoplasmic and exosome RNA fractionation experiment showing that circSPIRE1 mainly localized in the exosome. D Transmission electron microscopy of exosomes derived from original PDX and PDX LM. E Exosome marker TSG101 and CD63 analyzed through western blotting. F RT-PCR analysis of circSPIRE1 expression in HUVECs incubated with exosomes derived from p-CTRL and p-circSPIRE1 cells for 3, 6, 12, 24, and 48 h. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. G Effect of p-CTRL, p-circSPIRE1 and p-circSPIRE1 + Annexin V treatments on circSPIRE1 expression in HUVECs. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. H Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on tube formation ability of HUVECs by tube formation assay. Mean ± S.D. are provided (n = 3/group). Scale bar represents 100 µm. I Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on vascular outgrowth of rat aortic rings. Vascular outgrowth was quantified by counting all sprouts from one ring. Left, representative aortic ring images (n = 3/group). Data represent mean ± S.D.; dot plot reflects data points from three independent experiments. Scale bar represents 200 µm. J Permeability of the HUVEC monolayers to rhodamine–dextran (70 kDa) after exposure to exosomes derived from circSPIRE1 knockdown or overexpression cells for 72 h. Mean ± S.D. are shown. Dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. K Effect of circSPIRE1 knockdown or overexpression on tumor microvessel genesis. Top, representative tumor stained with CD34 anti-body. Bottom, microvessel density for each group. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. The P values were determined by Student’s t test. L VEGFR2, Occluding and Claudin5 protein expression in circSPIRE1 overexpression or circSPIRE1 overexpression combined GALNT3 knockdown HUVECs by western blot. M Schematic diagram of mechanism of circSPIRE1 during RCC metastasis in tumor microenvironment.CC BY · PubMed Central
Fig. 8 CS-EGCG NPs/circSPIRE1 overexpression plasmid nanocomplexes inhibits RCC metastasis in vivo. A Chemical structure of Chitosan-EGCG nanoparticles and schematic structures of CS-EGCG NPs complexing with plasmid. B Mean particle size of CS-EGCG nanoparticles. C TEM image of CS-EGCG NPs. D Binding ability of CS-EGCG NPs to circSPIRE1 overexpression plasmid at different mass ratios characterized by gel electrophoresis. E Cell cytotoxicity of CS-EGCG NPs complexing with plasmid assessed by CCK8 assay and the percentage of viable cells was calculated relative to untreated control cells. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. F Percentage of fluorescent cells analyzed by flow cytometry. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. G Fluorescence images of nude mice bearing metastatic renal cell carcinoma after tail vein injection of PBS, nude plasmid, lipo2000 and plasmid, as well as CS-EGCG NPs and overexpression plasmid complexes. H Top, bioluminescent in the lungs and tumor metastasis formed in the lungs after tail vein injection of PBS, nude plasmid, lipo2000, and plasmid, as well as CS-EGCG NPs and overexpression plasmid complexes. Bottom, statistical analysis of bioluminescent and tumor metastasis plots (n = 6 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test.
Fig. 8 CS-EGCG NPs/circSPIRE1 overexpression plasmid nanocomplexes inhibits RCC metastasis in vivo. A Chemical structure of Chitosan-EGCG nanoparticles and schematic structures of CS-EGCG NPs complexing with plasmid. B Mean particle size of CS-EGCG nanoparticles. C TEM image of CS-EGCG NPs. D Binding ability of CS-EGCG NPs to circSPIRE1 overexpression plasmid at different mass ratios characterized by gel electrophoresis. E Cell cytotoxicity of CS-EGCG NPs complexing with plasmid assessed by CCK8 assay and the percentage of viable cells was calculated relative to untreated control cells. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. F Percentage of fluorescent cells analyzed by flow cytometry. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. G Fluorescence images of nude mice bearing metastatic renal cell carcinoma after tail vein injection of PBS, nude plasmid, lipo2000 and plasmid, as well as CS-EGCG NPs and overexpression plasmid complexes. H Top, bioluminescent in the lungs and tumor metastasis formed in the lungs after tail vein injection of PBS, nude plasmid, lipo2000, and plasmid, as well as CS-EGCG NPs and overexpression plasmid complexes. Bottom, statistical analysis of bioluminescent and tumor metastasis plots (n = 6 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test.CC BY · PubMed Central

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Metadata

Authors
Shu, G., Lu, X., Pan, Y., Cen, J., Huang, K., Zhou, M., Lu, J., Dong, J., Han, H., Chen, W., Lin, J., Luo, J., & Zhang, J.
Venue
Oncogene
Publisher
Springer Science and Business Media LLC
Published
2023-04-12
Volume
42
Issue
22
Pages
1802-1820
ISSN
  • 0950-9232
  • 1476-5594
DOI
10.1038/s41388-023-02678-7
PMC
PMC10238271
Cited by
35
References
2
Language
en
Licence
Funders
  • National Natural Science Foundation of China (82073381, 81772514, 82002684, 81772718)
  • Pearl River S and T Nova Program of Guangzhou (201806010005)
  • Natural Science Foundation of Guangdong Province (S2019B151502046)
  • China Postdoctoral Science Foundation (236464, 2021TQ0381, 2021M703700)

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