KDM4A通过下调BMP9促进乳腺癌细胞MDA-MB-231的迁移和侵袭

doi:10.19401/j.cnki.1007-3639.2024.02.005

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

中图分类号:

R737.9

陈远香, 余涛, 杨诗雨, 曾涛, 魏兰, 张彦. KDM4A通过下调BMP9促进乳腺癌细胞MDA-MB-231的迁移和侵袭[J]. 中国癌症杂志, 2024, 34(2): 176-184.

CHEN Yuanxiang, YU Tao, YANG Shiyu, ZENG Tao, WEI Lan, ZHANG Yan. KDM4A promotes the migration and invasion of breast cancer cell line MDA-MB-231 by downregulating BMP9[J]. China Oncology, 2024, 34(2): 176-184.

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参考文献 26

[1]	HOUGHTON S C, HANKINSON S E. Cancer progress and priorities: breast cancer[J]. Cancer Epidemiol Biomarkers Prev, 2021, 30(5): 822-844. doi: 10.1158/1055-9965.EPI-20-1193
[2]	BARZAMAN K, KARAMI J, ZAREI Z, et al. Breast cancer: biology, biomarkers, and treatments[J]. Int Immunopharmacol, 2020, 84: 106535. doi: 10.1016/j.intimp.2020.106535
[3]	LANDSKRON G, DE LA FUENTE M, THUWAJIT P, et al. Chronic inflammation and cytokines in the tumor microenvironment[J]. J Immunol Res, 2014, 2014: 149185.
[4]	KONG R N, GAO J, JI L M, et al. Iguratimod ameliorates rheumatoid arthritis progression through regulating miR-146a mediated IRAK1 expression and TRAF6/JNK1 pathway: an in vivo and in vitro study[J]. Clin Exp Rheumatol, 2021, 39(2): 289-303. doi: 10.55563/clinexprheumatol/urhbn0
[5]	谭秋芬, 胡惠军. FEN1、GTF2IP23、KDM4A在乳腺癌组织中的表达研究[J]. 国际检验医学杂志, 2023, 44(3): 311-315.
	TAN Q F, HU H J. Expression and correlation of FEN1, GTF2IP23, and KDM4A in breast cancer tissues[J]. Int J Lab Med, 2023, 44(3): 311-315.
[6]	LEE J, THOMPSON J R, BOTUYAN M V, et al. Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor[J]. Nat Struct Mol Biol, 2008, 15(1): 109-111. doi: 10.1038/nsmb1326 pmid: 18084306
[7]	CHEN Y C, LIU X R, LI Y K, et al. Lung cancer therapy targeting histone methylation: opportunities and challenges[J]. Comput Struct Biotechnol J, 2018, 16: 211-223.
[8]	WANG G L, WEN B Q, DENG Z C, et al. Endothelial progenitor cells stimulate neonatal lung angiogenesis through FOXF1-mediated activation of BMP9/ACVRL1 signaling[J]. Nat Commun, 2022, 13(1): 2080. doi: 10.1038/s41467-022-29746-y pmid: 35440116
[9]	XU J Z, ZHOU Y M, ZHANG L L, et al. BMP9 reduces age-related bone loss in mice by inhibiting osteoblast senescence through Smad1-Stat1-P21 axis[J]. Cell Death Discov, 2022, 8(1): 254. doi: 10.1038/s41420-022-01048-8
[10]	HAN Y L, PAN Q Z, GUO Z X, et al. BMP9-induced vascular normalisation improves the efficacy of immunotherapy against hepatitis B virus-associated hepatocellular carcinoma[J]. Clin Transl Med, 2023, 13(5): e1247. doi: 10.1002/ctm2.1247 pmid: 37132170
[11]	CHEN H, NIO K, TANG H, et al. BMP9-ID1 signaling activates HIF-1α and VEGFA expression to promote tumor angiogenesis in hepatocellular carcinoma[J]. Int J Mol Sci, 2022, 23(3): 1475. doi: 10.3390/ijms23031475
[12]	YU H M, CHEN Y X, LANG L, et al. BMP9 promotes autophagy and inhibits migration and invasion in breast cancer cells through the c-Myc/SNHG3/mTOR signaling axis[J]. Tissue Cell, 2023, 82: 102073. doi: 10.1016/j.tice.2023.102073
[13]	WANG W, WENG Y G, REN W, et al. Biological roles of human bone morphogenetic protein 9 in the bone microenvironment of human breast cancer MDA-MB-231 cells[J]. Am J Transl Res, 2015, 7(9): 1660-1674. pmid: 26550465
[14]	WANG T, ZHANG Z H, WANG K, et al. Inhibitory effects of BMP9 on breast cancer cells by regulating their interaction with pre-adipocytes/adipocytes[J]. Oncotarget, 2017, 8(22): 35890-35901. doi: 10.18632/oncotarget.16271 pmid: 28415788
[15]	REN W, SUN X X, WANG K, et al. BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression[J]. Mol Biol Rep, 2014, 41(3): 1373-1383. doi: 10.1007/s11033-013-2982-8 pmid: 24413988
[16]	LI S, DAI H Y, HE Y, et al. BMP9 inhibits the growth of breast cancer cells by downregulation of the PI3K/Akt signaling pathway[J]. Oncol Rep, 2018, 40(3): 1743-1751. doi: 10.3892/or.2018.6572 pmid: 30015950
[17]	OUARNÉ M, BOUVARD C, BONEVA G, et al. BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer[J]. J Exp Clin Cancer Res, 2018, 37(1): 209. doi: 10.1186/s13046-018-0885-1 pmid: 30165893
[18]	NEVES REBELLO ALVES L, DUMMER MEIRA D, POPPE MERIGUETI L, et al. Biomarkers in breast cancer: An old story with a new end[J]. Genes, 2023, 14(7): 1364. doi: 10.3390/genes14071364
[19]	RANDO O J, CHANG H Y. Genome-wide views of chromatin structure[J]. Annu Rev Biochem, 2009, 78: 245-271. doi: 10.1146/annurev.biochem.78.071107.134639 pmid: 19317649
[20]	WEI C, DENG X G, GAO S D, et al. Cantharidin inhibits proliferation of liver cancer by inducing DNA damage via KDM4A-dependent histone H3K36 demethylation[J]. Evid Based Complement Alternat Med, 2022, 2022: 2197071.
[21]	SUN S S, YANG F J, ZHU Y C, et al. RETRACTED: KDM4A promotes the growth of non-small cell lung cancer by mediating the expression of Myc via DLX5 through the Wnt/β-catenin signaling pathway[J]. Life Sci, 2020, 262: 118508. doi: 10.1016/j.lfs.2020.118508
[22]	CHEN M, JIANG Y H, SUN Y B. KDM4A-mediated histone demethylation of SLC7A11 inhibits cell ferroptosis in osteosarcoma[J]. Biochem Biophys Res Commun, 2021, 550: 77-83. doi: 10.1016/j.bbrc.2021.02.137
[23]	XIONG J, NIE M F, FU C, et al. Hypoxia enhances HIF1α transcription activity by upregulating KDM4A and mediating H3K9me3, thus inducing ferroptosis resistance in cervical cancer cells[J]. Stem Cells Int, 2022, 2022: 1608806.
[24]	LI Y, WANG Y N, XIE Z, et al. JMJD2A facilitates growth and inhibits apoptosis of cervical cancer cells by downregulating tumor suppressor miR-491-5p[J]. Mol Med Rep, 2019, 19(4): 2489-2496.
[25]	LIN X, QIU W X, XIAO Y Y, et al. MiR-199b-5p suppresses tumor angiogenesis mediated by vascular endothelial cells in breast cancer by targeting ALK1[J]. Front Genet, 2019, 10: 1397. doi: 10.3389/fgene.2019.01397 pmid: 32082362
[26]	REN W, LIU Y H, WAN S H, et al. BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways[J]. PLoS One, 2014, 9(5): e96816. doi: 10.1371/journal.pone.0096816

Primer	Sequence (5'→3')
siRNA negative control	F: UUCUCCGAACGUGUCACGUTT
siRNA negative control	R: ACGUGACACGUUCGGAGAATT
siKDM4A	F: CCGAGUUUGUCUUGAAAUATT
siKDM4A	R: UAUUUCAAGACAAACUCGGTT
BMP9	F: CGTCCAACATTGTGCGGAG
BMP9	R: GACAGGAGACATAGAGTCGGAG
KDM4A	F: CCTCACTGCGCTGTCTGTAT
KDM4A	R: CCAGTCGAAGTGAAGCACAT
GAPDH	F: CAGCGACACCCACTCCTC
GAPDH	R: TGAGGTCCACCACCCTGT