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CRISPR/Cas9 regulates EphA2-CDH1 axis to inhibit the migration of castration-resistant prostate cancer cells

ABSTRACT: We ued CRISPR/Cas9 to knock out EphA2 gene in PC3 cells, and overexpress EphA2 gene in Du145 cells with overexpressed plasmids to explore the underlying mechanisms of EphA2 function in castration-resistant prostate cancer(CRPC) and clinical relevance, providing more evidence for EphA2 as a potential therapeutic target for CRPC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE267508 | GEO | 2024/05/20

REPOSITORIES: GEO

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Project description:Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis. Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

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CRISPR/Cas9 regulates EphA2-CDH1 axis to inhibit the migration of castration-resistant prostate cancer cells

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