[摘 要] 目的：探究Wnt5a通过上调miR-141-3p表达对前列腺癌（PCa）细胞血管生成拟态（VM）形成及肿瘤干细胞（CSC）特 性的影响。方法：选用人前列腺上皮细胞RWPE-1及PCa细胞PC-3、LNCaP和DU145，qPCR法检测细胞中miR-141-3p的表达， WB法检测细胞中Wnt5a蛋白的水平。通过质粒转染分别构建稳定敲减Wnt5a或miR-141-3p的LNCaP和DU145细胞株。采用 三维培养实验检测细胞形成VM的能力，CCK-8法检测细胞增殖能力及药物敏感性，划痕实验、Transwell实验检测细胞迁移和侵 袭能力，qPCR和WB法检测VM相关分子及CSC标志物的表达水平，克隆形成实验检测细胞克隆形成能力，流式细胞术分选并 计算CD133+ 细胞比例，qPCR和WB法检测CD133+ 细胞和CD133–细胞中miR-141-3p和Wnt5a的表达水平。通过质粒转染PCa 细胞构建稳定过表达Wnt5a的细胞株，qPCR法和双萤光素酶报告基因实验检测 Wnt5a 及不同 Wnt 下游信号通路抑制剂对 miR-141-3p表达及启动子活性的影响；通过转染si-c-Jun敲减Wnt5a过表达细胞中c-Jun的表达，qPCR法、双萤光素酶报告基因实 验及染色质免疫共沉淀实验验证c-Jun与miR-141-3p启动子的靶向结合关系。结果：miR-141-3p和Wnt5a在PCa细胞中的表达 显著高于RWPE-1细胞，在DU145细胞中相对表达量最高，在LNCaP细胞中相对表达量最低（P < 0.001）。下调Wnt5a或miR-141-3p 表达，均能显著抑制PCa细胞的VM形成能力及干细胞特性，显著抑制PCa细胞的增殖、迁移、侵袭能力，并能提高其对比卡鲁胺 的敏感性（P < 0.05或P < 0.01或P < 0.001）。下调Wnt5a的表达能够显著抑制miR-141-3p的表达及启动子转录活性（P < 0.01或 P < 0.05），上调Wnt5a的表达能够显著促进miR-141-3p的表达及启动子转录活性（P < 0.01或P < 0.001）；Wnt5a对miR-141-3p表 达及启动子转录活性的促进作用可被JNK/c-Jun通路抑制剂所抑制（P > 0.05）。下调c-Jun的表达能够显著抑制Wnt5a对miR-141-3p 表达及启动子转录活性的促进作用（P > 0.05）。c-Jun能够与miR-141-3p启动子的-348至-295序列结合，该片段缺失后，Wnt5a 无法促进miR-141-3p的表达（P > 0.05）。结论：Wnt5a/JNK/c-Jun信号通路能够上调miR-141-3p的表达，从而促进PCa细胞的 VM形成，其机制可能与CSC特性的激活有关。

[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.

[基金项目] 新疆维吾尔自治区自然科学基金青年基金项目（No. 2024D01C289）；新疆维吾尔自治区人民医院院内科研项目 （No. 20210105，No. 20200309）