超级增强子介导GSE1的转录并通过Wnt/β-catenin信号通路促进乳腺癌细胞增殖的机制

doi:10.3969/j.issn.1006-5725.2025.18.003

摘要/Abstract

摘要：

目的研究超级增强子（super enhancer，SE）调控基因抑制因子1（genetic suppressor element 1， GSE1）表达，及其对乳腺癌细胞MCF-7增殖能力的影响及分子机制。方法使用GEO数据库中MCF-7细胞的ChIP-seq数据集筛选SE及其驱动的癌基因GSE1；HPA和TCGA数据库分别分析GSE1在泛癌和不同乳腺癌亚型中的表达；GEPIA数据库分析GSE1高表达分组和低表达分组之间乳腺癌患者的总体生存期（overall survival，OS）；免疫组织化学染色（immunohistochemistry， IHC）分析GSE1在乳腺癌组织中的表达情况；RT-qPCR验证GSE1在不同乳腺癌细胞中mRNA的表达；使用溴结构域蛋白4（bromodomain-containing protein 4，BRD4）抑制剂JQ1处理MCF-7细胞；构建GSE1干扰载体，进行MCF-7细胞株转染，Western blot评估GSE1干扰表达的效率，通过CCK-8、集落形成实验、划痕愈合实验和Transwell迁移实验评估细胞增殖和迁移情况；String数据库分析GSE1互作蛋白；Western blot检测上皮-间充质转化（epithelial-mesenchymal transition，EMT）和Wnt/β-catenin信号通路相关蛋白变化情况。结果基因组浏览器（integrative genomics viewer，IGV）可视化MCF-7细胞的ChIP-seq数据，明确GSE1作为候选基因；结合Rank Ordering Of Super Enhancers（ROSE）算法预测GSE1的SE区域在MCF-7细胞基因组的位置；使用BRD4抑制剂JQ1处理MCF-7细胞后，GSE1和BRD4表达受到抑制（P < 0.05）；HPA、TCGA、GEPIA数据库以及IHC分析显示GSE1在乳腺癌中高表达并与乳腺癌患者较差的预后生存期相关（P < 0.05）；RT-qPCR结果显示GSE1 mRNA在乳腺癌细胞中的表达高于正常乳腺上皮细胞（P < 0.05）；String数据库分析显示GSE1与Snail（Snai1）密切相关；与对照组相比转染GSE1干扰载体后抑制乳腺癌细胞MCF-7的增殖和迁移，且上皮相关蛋白E-cadherin、Occludin表达升高，而间充质相关蛋白N-cadherin、Snail表达降低（P < 0.05）；与对照组相比敲低GSE1， Wnt/β-catenin信号通路相关蛋白β-catenin、Wnt-5a、Cyclin-D1表达降低，Axin1蛋白表达升高（P < 0.05）。结论 SE驱动GSE1并通过Wnt/β-catenin信号通路诱导乳腺癌细胞MCF-7的EMT过程，进而促进增殖和迁移。

关键词: 超级增强子, 基因抑制因子1, 上皮-间充质转化, 增殖, 迁移, Wnt/β-catenin信号通路

Abstract:

Objective To investigate the molecular mechanism by which super enhancers （SEs） regulate the expression of the genetic suppressor element 1 （GSE1） and influence the proliferation of breast cancer cell line MCF-7. Methods The oncogene GSE1， driven by SEs， was identified through analysis of the ChIP-seq dataset of MCF-7 cells obtained from the GEO database. The protein expression level of GSE1 was assessed via Western blot following treatment with the bromodomain-containing protein 4 （BRD4） inhibitor JQ1. The expression of GSE1 across various cancers and different breast cancer subtypes was analyzed using the Human Protein Atlas （HPA） and The Cancer Genome Atlas （TCGA） databases， respectively. Overall survival （OS） of breast cancer patients was compared between the GSE1 high-expression and low-expression groups using the GEPIA database. Immunohistochemistry （IHC） was performed to evaluate GSE1 expression in breast cancer tissue samples. The mRNA expression levels of GSE1 in different breast cancer cell lines were validated by RT-qPCR. A GSE1 interference vector was constructed and transfected into MCF-7 cells， and the knockdown efficiency was assessed using Western blot. Cell proliferation and migration were evaluated using CCK-8 assay， colony formation assay， wound healing assay， and Transwell migration assay. Potential GSE1-interacting proteins were predicted using the STRING database. Finally， Western blot analysis was conducted to assess changes in epithelial-mesenchymal transition （EMT）-related proteins and key components of the Wnt/β-catenin signaling pathway. Results The integrative genomics viewer （IGV） was used to visualize ChIP-seq data from MCF-7 cells， and the rank ordering of super enhancers （ROSE） algorithm was applied to predict the SE region of GSE1 in the MCF-7 genome. GSE1 and BRD4 expression levels were significantly reduced following JQ1 treatment （P < 0.05）. High expression levels of GSE1 in breast cancer were confirmed through analyses using the HPA， TCGA， and GEPIA databases， as well as IHC， and these findings were associated with poor prognostic outcomes in breast cancer patients （P < 0.05）. RT-qPCR results further demonstrated that GSE1 is significantly upregulated in breast cancer cells （P < 0.05）. Analysis of the STRING database revealed a strong correlation between GSE1 and Snail （Snai1）. Transfection of a GSE1-specific interference vector significantly inhibited the proliferation and migration of MCF-7 cells compared to the control group， upregulated E-cadherin and Occludin expression， and downregulated N-cadherin and Snail expression （P < 0.05）. Additionally， knockdown of GSE1 resulted in decreased expression of Wnt/β-catenin signaling pathway-related proteins， including β-catenin， Wnt-5a， and Cyclin-D1， along with increased Axin1 expression （P < 0.05）. Conclusion SE driven GSE1 promotes the proliferation， migration and EMT of MCF-7 cells via the Wnt/β-catenin signaling pathway.

Key words: super-enhancer, GSE1, epithelial-mesenchymal transition, proliferation, migration, Wnt/β-catenin signaling pathway

中图分类号:

R737.9

杜沛静,虎恩喜,陶翔,曹佳,王立斌. 超级增强子介导GSE1的转录并通过Wnt/β-catenin信号通路促进乳腺癌细胞增殖的机制[J]. 实用医学杂志, 2025, 41(18): 2796-2805.

Peijing DU,Enxi HU,Xiang TAO,Jia CAO,Libin WANG. Super⁃enhancer⁃mediated GSE1 activation drives breast cancer proliferation through Wnt/β⁃catenin signaling[J]. The Journal of Practical Medicine, 2025, 41(18): 2796-2805.

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