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山东大学学报 (医学版) ›› 2023, Vol. 61 ›› Issue (1): 17-26.doi: 10.6040/j.issn.1671-7554.0.2022.1064

• 基础医学 • 上一篇    

长链非编码RNA ZNF528-AS1促进乳腺癌他莫昔芬耐药及进展转移

张建树1,张瀚文1,赵文静2   

  1. 山东大学齐鲁医院 1. 乳腺外科;2.生物样本资源库, 山东 济南 250012
  • 发布日期:2023-01-10
  • 通讯作者: 赵文静. E-mail:zhaowenjing0911@163.com
  • 基金资助:
    中国博士后科学基金(2017M612292)

LncRNA ZNF528-AS1 promotes tamoxifen resistance and progression of breast cancer

ZHANG Jianshu1, ZHANG Hanwen1, ZHAO Wenjing2   

  1. 1. Department of Breast Surgery;
    2. Pathology Tissue Bank, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China
  • Published:2023-01-10

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摘要： 目的 探讨长链非编码RNA ZNF528-AS1对乳腺癌他莫昔芬耐药及进展转移的影响及潜在调控机制。 方法 利用RNA-seq高通量测序技术分析乳腺癌他莫昔芬耐药细胞/成瘤组织与亲本细胞/成瘤组织间差异表达的lncRNA,筛选与乳腺癌他莫西芬耐药相关的候选lncRNA,利用实时定量PCR(qRT-PCR)技术验证候选lncRNA的表达。通过脂质体转染乳腺癌细胞,构建过表达及敲低ZNF528-AS1的细胞模型。利用细胞增殖实验、克隆形成实验、成球实验、5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)实验检测ZNF528-AS1对乳腺癌细胞增殖、肿瘤干性及他莫昔芬耐药性的影响。Transwell迁移和侵袭实验检测ZNF528-AS1对乳腺癌细胞转移能力的影响。利用RNA-seq高通量测序及生物信息学分析技术寻找ZNF528-AS1的下游调控通路,并通过Western blotting及细胞功能实验等进行验证。 结果 RNA-seq高通量测序分析筛选出了在他莫昔芬耐药细胞/成瘤组织与亲本细胞/成瘤组织间差异表达的lncRNAs。qRT-PCR实验结果证实了差异lncRNA的表达,并发现ZNF528-AS1在他莫昔芬耐药细胞中的表达上调最为明显。在乳腺癌细胞中过表达ZNF528-AS1能够提高细胞的增殖水平、克隆形成能力、肿瘤干性及对他莫昔芬的耐药性。此外,过表达ZNF528-AS1可增强乳腺癌细胞的迁移及侵袭能力。相应地,在乳腺癌细胞中敲低ZNF528-AS1能够降低细胞的增殖、他莫昔芬耐药性、肿瘤干性、迁移及侵袭等能力。RNA-seq高通量测序结合生物信息学分析结果显示,过表达ZNF528-AS1能够参与调控转化生长因子-β(TGF-β)信号通路。Western blotting结果证实ZNF528-AS1能够同时激活经典和非经典TGF-β信号通路。TGF-β通路抑制剂SB431542能够逆转ZNF528-AS1过表达增强的细胞增殖、迁移及侵袭能力。 结论 ZNF528-AS1能够促进乳腺癌他莫昔芬耐药及进展转移,其作用机制可能与激活TGF-β通路有关。

关键词: 乳腺癌, ZNF528-AS1, 他莫昔芬耐药, 肿瘤干性, 进展转移

Abstract: Objective To investigate the effects of long non-coding RNA(LncRNA)ZNF528-AS1 on tamoxifen resistance and progression of breast cancer as well as the potential regulatory mechanism. Methods The differentially expressed lncRNAs among tamoxifen-resistant breast cancer cells/xenograft tissues and parent cells/ xenograft tissues were analyzed with RNA-seq high-throughput sequencing technology, and candidate lncRNAs associated with tamoxifen resistance were screened. The expressions of candidate lncRNAs were verified with quantitative real-time PCR(qRT-PCR). Breast cancer cells were transfected with liposomes to construct ZNF528-AS1 overexpression and knockdown cell models. The effects of ZNF528-AS1 on the proliferation, tumor stemness and tamoxifen resistance were detected with MTT, colony formation, mammosphere formation, and EdU assays. The role of ZNF528-AS1 in the metastasis of breast cancer was evaluated with Transwell migration and invasion assays. The downstream regulatory pathways of ZNF528-AS1 were detected with RNA-seq high-throughput sequencing and bioinformatics analysis, and verified with Western blotting and functional experiments. Results The differentially expressed lncRNAs were screened out and verified. ZNF528-AS1 was significantly upregulated in tamoxifen-resistant cells. Overexpression of ZNF528-AS1 in breast cancer cells promoted cell proliferation, colony formation ability, tumor stemness, and tamoxifen resistance. In addition, ZNF528-AS1 overexpression enhanced the migration and invasion abilities of breast cancer cells. ZNF528-AS1 knockdown reduced cell proliferation, tamoxifen resistance, tumor stemness, migration, and invasion. ZNF528-AS1 overexpression might be involved in the regulation of transforming growth factor(TGF-β)signaling pathway. ZNF528-AS1 activated both canonical and non-canonical TGF-β signaling pathways. TGF-β pathway inhibitor SB431542 reversed the proliferation, migration and invasion of breast cancer cells enhanced by ZNF528-AS1 overexpression. Conclusion ZNF528-AS1 can promote tamoxifen resistance and progression of breast cancer, and its mechanism might be related to the activation of TGF-β pathway.

Key words: Breast cancer, ZNF528-AS1, Tamoxifen resistance, Tumor stemness, Progression

中图分类号: 

  • R737.9
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