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Archive > Volume 32 Issue 10 > 2025,32(10):1010-1018. DOI:10.3872/j.issn.1007-385X.2025.10.002 Prev Next
Basic Research
Wnt5a promotes vasculogenic mimicry and stemness in prostate cancer cells through miR-141-3p upregulation
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Abstract:
[Abstract] Objective: To investigate the effects of Wnt5a on the vasculogenic mimicry (VM) and cancer stem cell (CSC) properties of prostate cancer (PCa) cells by upregulating the expression of miR-141-3p. Methods: Human prostate epithelial cell line RWPE-1 and PCa cell lines PC-3, LNCaP, and DU145 were cultured. qPCR was employed to detect miR-141-3p expression, and Western blotting (WB) was used to measure Wnt5a protein levels. Stable Wnt5a-knockdown or miR-141-3p-knockdown LNCaP and DU145 cell lines were established respectively via plasmid transfection. VM formation ability was assessed by three-dimensional culture assay. Cell proliferation ability and drug sensitivity were measured by CCK-8 assay. Cell migration and invasion abilities were detected using wound healing and Transwell assays, respectively. The expressions of VM-related molecules and CSC markers were detected by qPCR and WB. Colony formation ability was determined by clonogenic assay. The proportion of CD133+ cells was sorted and calculated by flow cytometry. The expressions of miR-141-3p and Wnt5a in CD133+ and CD133– cells were detected by qPCR and WB. Stable Wnt5a-overexpressing PCa cell lines were constructed via plasmid transfection. The effects of Wnt5a and different Wnt pathway downstream inhibitors on miR-141-3p expression and promoter activity were detected by qPCR and dual-luciferase reporter assays. Expression of c-Jun was knocked down in Wnt5a-overexpressing cells using si-c-Jun transfection. The target binding relationship between c-Jun and the miR-141-3p promoter was verified by qPCR, dual-luciferase reporter assay, and chromatin immunoprecipitation assay. Results: The expressions of miR-141-3p and Wnt5a were significantly higher in PCa cells compared with those in RWPE-1 cells, with the highest relative expression in DU145 cells and the lowest in LNCaP cells (P < 0.001). Downregulation of Wnt5a or miR-141-3p significantly inhibited VM formation ability and stemness of PCa cells, and significantly suppressed the proliferation, migration, invasion abilities, and enhanced the sensitivity to bicalutamide of PCa cells (P < 0.05 or P < 0.01 or P < 0.001). Downregulation of Wnt5a significantly inhibited miR-141-3p expression and promoter transcriptional activity (P < 0.01 or P < 0.05), whereas upregulation of Wnt5a significantly promoted miR-141-3p expression and promoter transcriptional activity (P < 0.01 or P < 0.001). The promoting effect of Wnt5a on miR-141-3p expression and promoter transcriptional activity could be inhibited by a JNK/c-Jun pathway inhibitor (P > 0.05). Downregulation of c-Jun significantly inhibited the promoting effect of Wnt5a on miR-141-3p expression and promoter transcriptional activity (P > 0.05). c-Jun could bind to the -348 to -295 sequence of the miR-141-3p promoter. In absence of this fragment Wnt5a wouldn’t promote miR-141-3p expression (P > 0.05). Conclusion: The Wnt5a/JNK/c-Jun signaling pathway can upregulate miR-141-3p expression, and thereby promote VM formation in PCa cells, possibly by activating CSC properties.
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