KDM4A通过下调BMP9促进乳腺癌细胞MDA-MB-231的迁移和侵袭

陈远香, 余涛, 杨诗雨, 曾涛, 魏兰, 张彦

  1. 重庆医科大学检验医学院临床检验诊断学教育部重点实验室，重庆 400016
  • 收稿日期:2023-09-19 修回日期:2023-12-18 出版日期:2024-02-29 发布日期:2024-03-14
  • 通信作者: 张彦
  • 作者简介:陈远香（ORCID: 0009-0004-4691-9984），硕士。
  • 基金资助:
    国家自然科学基金(81974449)

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摘要/Abstract

摘要：

背景与目的：外源性骨形态发生蛋白9（bone morphogenetic protein 9，BMP9）能抑制人乳腺癌的恶性进展，但其在乳腺癌中常异常低表达。本研究拟探索表观遗传修饰的组蛋白赖氨酸特异性去甲基化酶4A（lysine specific demethylase 4A，KDM4A）在乳腺癌中的表达及作用，探究KDM4A与BMP9之间的关系及其可能的调节机制。方法：通过生物信息学方法分析KDM4A在乳腺癌中的表达及其与BMP9之间的关系，采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）和蛋白质印迹法（Western blot）进行验证；采用染色质免疫沉淀（chromatin immunoprecipitation，ChIP）验证KDM4A对BMP9的调控作用，采用RNA稳定性实验及CHX蛋白稳定性实验验证KDM4A对BMP9表达的影响。采用RNA干扰技术及敲低BMP9的腺病毒构建转染KDM4A小干扰RNA（siKDM4A）或感染siBMP9腺病毒（Ad-siBMP9）的外源性重组MDA-MB-231细胞，采用划痕愈合实验、transwell实验分别检测细胞迁移及侵袭能力。结果：生物信息学分析结果表明，KDM4A在乳腺癌中的表达明显高于正常组织，KDM4A与BMP9在乳腺癌中的表达呈负相关关系。RTFQ-PCR及Western blot结果显示，KDM4A在不同乳腺癌细胞系中均高表达，敲低KDM4A可显著上调BMP9。ChIP实验证实，KDM4A可显著富集于BMP9基因启动子区域，降低其组蛋白赖氨酸36号位而不是4号位甲基化水平，从而沉默BMP9表达。RNA稳定性实验及CHX蛋白稳定性实验证实，KDM4A对BMP9的mRNA表达无明显影响，但可影响其蛋白降解。敲低KDM4A后，乳腺癌细胞MDA-MB-231的迁移及侵袭能力均受到明显抑制，而这种作用可被敲低BMP9所部分逆转。结论：KDM4A在乳腺癌组织及乳腺癌细胞MDA-MB-231中高表达，并可通过下调BMP9基因启动子区域组蛋白甲基化水平沉默其表达，以及在蛋白水平而不是mRNA水平影响BMP9稳定性，促进乳腺癌的迁移和侵袭。

关键词: 乳腺癌, 赖氨酸特异性去甲基化酶4A, 组蛋白去甲基化, 骨形态发生蛋白9

Abstract:

Background and purpose: Exogenous bone morphogenetic protein 9 (BMP9) inhibits the malignant progression of human breast cancer, but its expression is often abnormally low in breast cancer. In this study, we intended to explore the expression and role of epigenetically-modified histone lysine-specific demethylase 4A (KDM4A) in breast cancer, and to investigate the relationship between KDM4A and BMP9 and its possible regulatory mechanism. Methods: The expression of KDM4A in breast cancer and its relationship with BMP9 were analyzed by bioinformatics and verified by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. Chromatin immunoprecipitation (ChIP) verified the regulatory role of KDM4A on BMP9, and RNA stability experiments and CHX protein stability experiments verified the effect of KDM4A in BMP9 expression. Exogenous recombinant MDA-MB-231 cells transfected with KDM4A small interfering RNA (siKDM4A) or infected with siBMP9 adenovirus (Ad-siBMP9) were constructed using RNA interference technology and adenoviruses knocking down BMP9, and the migratory and invasive abilities of the cells were detected by scratch healing assay and transwell assay, respectively. Results: Bioinformatics analysis showed that the expression of KDM4A was significantly higher in breast cancer than in normal tissues, and there was a negative correlation between the expression of KDM4A and that of BMP9 in breast cancer; RTFQ-PCR and Western blot showed that KDM4A was highly expressed in different breast cancer cell lines, and the knockdown of KDM4A significantly up-regulated BMP9. ChIP experiment confirmed that KDM4A could be significantly enriched in the promoter region of BMP9 gene, reducing its histone lysine 36 position instead of position 4 methyl status, thus silencing the expression of BMP9. RNA stability assay and CHX protein stability assay confirmed that KDM4A had no significant effect on the mRNA of BMP9, but could affect its protein degradation. After knocking down KDM4A, the migration and invasion abilities of breast cancer cells MDA-MB-231 were significantly inhibited, and this effect could be partially reversed by knocking down BMP9. Conclusion: KDM4A is highly expressed in breast cancer and breast cancer cell MDA-MB-231, and can silence its expression by down-regulating the level of histone methylation in the promoter region of the BMP9 gene, as well as affecting the stability of BMP9 at the protein level rather than at the level of mRNA, and promoting the migration and invasion of breast cancer.

Key words: Breast cancer, Lysine-specific demethylase 4A, Histone demethylation, Bone morphogenetic protein 9

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