毛兰素通过阻断Wnt/β-catenin信号抑制乳腺癌细胞增殖和迁移

doi:10.12122/j.issn.1673-4254.2026.04.12

摘要/Abstract

摘要：

目的探讨毛兰素对乳腺癌细胞的增殖、迁移、侵袭和凋亡的影响及潜在的分子机制。方法分别用0、12.5、25、50、100 nmol/L毛兰素处理乳腺癌细胞系T-47D和MCF-7，采用CCK-8法检测12、24、36、48、72 h的细胞活力；克隆形成、细胞划痕、Transwell侵袭和细胞衰老实验分别检测毛兰素对T-47D和MCF-7细胞增殖、迁移、侵袭能力以及细胞衰老的影响。通过毛兰素mRNA微阵列以及Enrichr数据库探索毛兰素的生物学功能。通过流式细胞术检测不同浓度毛兰素对T-47D和MCF-7细胞凋亡率的影响。通过Western blotting检测细胞凋亡、上皮-间充质转化（EMT）以及Wnt/β-catenin通路相关蛋白的表达情况。结果毛兰素呈浓度依赖性抑制T-47D和MCF-7细胞活力、增殖、迁移与侵袭能力，并促进细胞衰老（P<0.05）。毛兰素相关mRNA微阵列分析结果显示，共有1064个差异表达基因（DEGs）（P<0.05），其中948个表达上调基因，116个表达下调基因。GO富集分析结果显示，毛兰素的DEGs主要富集在EMT的调节、含胶原蛋白的细胞外基质及钙离子结合等功能中；KEGG和WikiPathways富集分析结果显示，毛兰素可能通过调节PI3K-Akt信号通路、Wnt/β-catenin信号通路以及细胞凋亡影响乳腺癌的发生与进展。流式细胞术结果显示，毛兰素呈浓度依赖性诱导乳腺癌细胞凋亡（P<0.05）。Western blotting结果显示，随着毛兰素浓度升高，促凋亡蛋白Bax、Caspase3表达上升，而抗凋亡蛋白Bcl-2表达下降（P<0.05）；EMT相关蛋白Snail、N-cadherin、β-catenin表达下降（P<0.05）；Wnt/β-catenin信号相关蛋白TCF4、Cyclin D1、c-Myc表达下降（P<0.05）；与100 nmol/L毛兰素组相比，100 nmol/L毛兰素+Wnt/β-catenin激动剂组TCF4、Cyclin D1、c-Myc表达增加（P<0.05）。结论毛兰素能抑制乳腺癌细胞增殖、迁移和侵袭，并诱导细胞衰老和凋亡，其分子机制可能与阻断Wnt/β-catenin信号通路，协同诱导细胞凋亡并逆转EMT过程有关。

关键词: 毛兰素, 乳腺癌, 上皮-间充质转化, Wnt/β-catenin信号通路

Abstract:

Objective To investigate the effect of erianin on proliferation, migration, invasion, and apoptosis of breast cancer cells and the underlying mechanisms. Methods Breast cancer cell lines T-47D and MCF-7 treated with 0, 12.5, 25, 50, and 100 nmol/L erianin for 12, 24, 36, 48, and 72 h were examined for cell viability using CCK-8 assay. The effects of erianin on cell proliferation, migration, invasion, senescence and apoptosis were evaluated using clone formation, wound healing, Transwell invasion, and senescence assays and flow cytometry. mRNA microarray analysis and the Enrichr database were used to explore the biological functions of erianin. Western blotting was used to detect the changes in protein expressions related to apoptosis, epithelial-mesenchymal transition (EMT), and the Wnt/β-catenin pathway. Results Erianin concentration-dependently inhibited cell viability, proliferation, migration, and invasion, and promoted senescence in T-47D and MCF-7 cells. Microarray analysis identified 1064 differentially expressed genes (DEGs), including 948 upregulated and 116 downregulated genes, which were involved primarily in EMT regulation, collagen-containing extracellular matrix, calcium ion binding, the PI3K-Akt signaling pathway, the Wnt/β-catenin signaling pathway, and apoptosis. Flow cytometry confirmed that erianin concentration-dependently induced apoptosis in the breast cancer cells, upregulated the expressions of Bax and caspase-3, decreased Bcl-2 expression, and lowered the expressions of EMT-related proteins (Snail, N-cadherin, and β‑catenin) and Wnt/β‑catenin signaling proteins (TCF4, Cyclin D1, and c-Myc). In the breast cancer cells treated with 100 nmol/L erianin, the application of a Wnt/β‑catenin agonist significantly increased the proteins expressions of TCF4, Cyclin D1, and c-Myc. Conclusion Erianin inhibits proliferation, migration, and invasion and induces senescence and apoptosis in breast cancer cells possibly by suppressing the Wnt/β-catenin signaling pathway to induce cell apoptosis and reverse EMT of the cells.

Key words: erianin, breast cancer, epithelial to mesenchymal transition, Wnt/β-catenin signaling pathway

范东伟, 李煊赫, 姚廷敬, 金从稳, 王兴亮, 黄诚. 毛兰素通过阻断Wnt/β-catenin信号抑制乳腺癌细胞增殖和迁移[J]. 南方医科大学学报, 2026, 46(4): 838-847.

Dongwei FAN, Xuanhe LI, Tingjing YAO, Congwen JIN, Xingliang WANG, Cheng HUANG. Erianin inhibits proliferation and migration of breast cancer cells in vitro by inhibiting Wnt/β-catenin signaling[J]. Journal of Southern Medical University, 2026, 46(4): 838-847.

图/表 11

图1 毛兰素对T-47D和MCF-7细胞活力的影响

Fig.1 Effect of erianin on viability of T-47D and MCF-7 cells. A: CCK-8 assay results of T-47D cells treated with different concentrations of erianin for 12, 24, 36, 48, and 72 h. B: CCK-8 assay results of MCF-7 cells treated with different concentrations of erianin for 12, 24, 36, 48, and 72 h. Data are presented as Mean±SD (n=6). *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol/L.

图2 毛兰素对T-47D和MCF-7细胞增殖能力的影响

Fig.2 Effect of erianin on proliferative capacity of T-47D and MCF-7 cells. A: Clone formation assay results of T-47D cells treated with different concentrations of erianin for 48 h. B: Clone formation assay results of MCF-7 cells treated with different concentrations of erianin for 48 h. Data are presented as Mean±SD (n=3). ***P<0.001 vs 0 nmol/L.

图3 毛兰素对T-47D和MCF-7细胞迁移能力的影响

Fig.3 Effect of erianin on migration of T-47D and MCF-7 cells. A: Wound healing assay results of T-47D cells treated with different concentrations of erianin for 48 h. B: Wound healing assay results of MCF-7 cells treated with different concentrations of erianin for 48 h. Scale bar=200 μm. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol/L.

图4 毛兰素对T-47D和MCF-7细胞侵袭能力的影响

Fig.4 Effect of erianin on invasion of T-47D and MCF-7 cells. A: Transwell invasion assay results of T-47D cells treated with different concentrations of erianin for 48 h. B: Transwell invasion assay results of MCF-7 cells treated with different concentrations of erianin for 48 h. Data are presented as Mean±SD (n=3). ***P<0.001 vs 0 nmol/L.

图5 毛兰素对T-47D和MCF-7细胞衰老能力的影响

Fig.5 Effect of erianin on cellular senescence in T-47D and MCF-7 cells (Original magnification: ×200). A: Senescence-associated β-galactosidase staining results of T-47D cells treated with different concentrations of erianin for 48 h. B: Senescence-associated β-galactosidase staining results of MCF-7 cells treated with different concentrations of erianin for 48 h. Data are presented as Mean±SD (n=3). *P<0.05, ***P<0.001 vs 0 nmol/L.

图6 毛兰素处理T-47D细胞48 h后的DEGs火山图

Fig.6 Volcano plot of differentially expressed genes (DEGs) in T-47D cells treated with erianin for 48 h.

图7 毛兰素DEGs的生物学功能富集分析

Fig.7 Biological functional enrichment analysis of DEGs regulated by erianin. A: GO enrichment analysis of DEGs in breast cancer. B: KEGG and WikiPathways enrichment analysis of DEGs in breast cancer.

图8 毛兰素对T-47D和MCF-7细胞凋亡的影响

Fig.8 Effect of erianin on apoptosis of T-47D and MCF-7 cells. A: Apoptosis rate of T-47D cells treated with different concentrations of erianin for 48 h analyzed by flow cytometry. B: Apoptosis rate of MCF-7 cells treated with different concentrations of erianin for 48 h analyzed by flow cytometry. Data are presented as Mean±SD (n=3). ***P<0.001 vs 0 nmol/L.

图9 毛兰素对乳腺癌细胞中凋亡相关蛋白表达水平影响

Fig.9 Effect of erianin on expression levels of apoptosis-related proteins in breast cancer cells. A: Western blotting of Bax, caspase-3, and Bcl-2 expression in T-47D cells treated with erianin. B: Western blotting of Bax, caspase-3, and Bcl-2 expression in MCF-7 cells treated with erianin. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol/L.

图10 毛兰素对乳腺癌细胞中EMT标志蛋白表达水平影响

Fig.10 Effect of erianin on expression levels of EMT marker proteins in breast cancer cells. A: Western blotting of Snail, N-cadherin, and β-catenin expression in T-47D cells treated with erianin. B: Western blotting of Snail, N-cadherin, and β-catenin expression in MCF-7 cells treated with erianin. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol/L.

图11 毛兰素对乳腺癌细胞Wnt/β-catenin信号的影响

Fig.11 Effect of erianin on the Wnt/β-catenin signaling pathway in breast cancer cells. A: Western blotting of TCF4, Cyclin D1, and c-Myc expression in T-47D cells treated with erianin. B: Western blotting of TCF4, Cyclin D1, and c-Myc expression in MCF-7 cells treated with erianin. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs 0 nmol/L. ##P<0.01, ###P<0.001.

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