干扰MAD2L1基因表达对乳腺癌细胞凋亡的影响及机制

doi:10.6040/j.issn.1671-7554.0.2022.0282

摘要/Abstract

摘要： 目的探讨干扰有丝分裂阻滞缺陷2样蛋白1(MAD2L1)基因表达对乳腺癌(BC)细胞凋亡的影响及其机制。方法采用qRT-PCR检测正常乳腺上皮细胞系MCF-10A和4种BC细胞系(MDA-MB-231、MCF-7、SK-BR-3和BT-20)中MAD2L1 mRNA表达水平。将MAD2L1 siRNA转染至MDA-MB-231细胞中,qRT-PCR和Western blotting检测细胞中MAD2L1 mRNA和蛋白表达水平;MTT检测细胞增殖能力;流式细胞术法检测细胞凋亡率;Western blotting检测细胞中Bax、Bcl-2、cleaved-caspase-3、p-p38MAPK(Thr180/Thr182)和p38MAPK等蛋白表达水平。采用p38MAPK抑制剂SB203580联合处理上述细胞,Annexin V-FITC/PI法检测细胞凋亡率的变化;Western blotting检测Bax、Bcl-2、cleaved-caspase-3、p-p38MAPK和p38MAPK表达水平的变化。结果 BC细胞系中MAD2L1 mRNA表达水平高于MCF-10A细胞,其中MDA-MB-231细胞最为显著。干扰MAD2L1基因可降低MDA-MB-231细胞中MAD2L1 mRNA和蛋白表达水平(t_mRNA=10.51,P_mRNA<0.001;t_蛋白=18.30,P_蛋白<0.001),同时抑制细胞增殖(F_组别=243.36、F_时间=44.00、F_{组别×时间}=9.881,P均<0.001),促进细胞凋亡(t=9.10,P<0.001),并上调Bax、cleaved-caspase-3和p-p38MAPK蛋白表达水平(t_Bax=15.05,P_Bax<0.001;t_{cleaved-caspase-3}=5.26,P_{cleaved-caspase-3}=0.006;t_p-p38MAPK=28.46,P_p-p38MAPK<0.001),下调Bcl-2蛋白表达水平(t_Bcl-2=14.23,P<0.001)。然而,SB203580处理可抑制MAD2L1基因干扰对MDA-MB-231细胞凋亡的诱导作用(P=0.002)。结论干扰MAD2L1基因表达可抑制MDA-MB-231细胞增殖,并诱导其凋亡,其作用机制可能与激活p38MAPK信号通路有关。

关键词: 乳腺癌, 有丝分裂阻滞缺陷2样蛋白1, 细胞凋亡, p38MAPK信号通路

Abstract: Objective To explore the effects of interference with mitotic arrest deficient 2-like protein 1(MAD2L1)gene expression on breast cancer(BC)cell apoptosis and the mechanism. Methods The mRNA expressions of MAD2L1 in normal breast epithelial cell line MCF-10A and BC cell lines, including MDA-MB-231, MCF-7, SK-BR-3 and BT-20, were detected with qRT-PCR. After MAD2L1 siRNA was transfected into MDA-MB-231 cells, the mRNA and protein expressions of MAD2L1 were detected with qRT-PCR and Western blotting; cell proliferation ability was measured with MTT; cell apoptosis was detected with Annexin V-FITC/PI; expressions of Bax, Bcl-2, cleaved-caspase-3, p-p38MAPK(Thr180/Thr182)and p38MAPK were determined with Western blotting. After the above cells were treated with the p38MAPK inhibitor SB203580 in combination, the changes in apoptosis rate were detected with flow cytometry, and changes in the expressions of Bax, Bcl-2, cleaved-caspase-3, p-p38MAPK and p38MAPK were detected with Western blotting. Results The mRNA expression of MAD2L1 in BC cell line, especially in MDA-MB-231 cells, was significantly higher than that in MCF-10A cells. Interference with MAD2L1 gene reduced the mRNA and protein expressions of MAD2L1 in MDA-MB-231 cells(t_mRNA=10.51, P_mRNA<0.001; t_protein=18.30, P_protein<0.001), inhibited cell proliferation(F_group=243.36, F_time=44.00, F_{group × time}=9.881, all P<0.001), promoted cell apoptosis(t=9.10, P<0.001), up-regulated the protein expressions of Bax, cleaved-caspase-3 and p-p38MAPK(t_Bax=15.05, P_Bax<0.001; t_{cleaved-caspase-3}=5.26, P_{cleaved-caspase-3}=0.006; t_p-p38MAPK=28.46, P_p-p38MAPK<0.001), and down-regulated the protein expression of Bcl-2(t_Bcl-2=14.23, P_Bcl-2<0.001). However, SB203580 treatment inhibited the induction of apoptosis by MAD2L1 gene interference on MDA-MB-231 cells(P=0.002). Conclusion Interfering with the expression of MAD2L1 gene can inhibit the proliferation of MDA-MB-231 cells and induce apoptosis, and the mechanism may be related to the activation of the p38MAPK signaling pathway.

Key words: Breast cancer, Mitotic arrest defect 2-like protein 1, Apoptosis, p38MAPK signaling pathway

中图分类号:

R737.9

封海岗,刘国文,曹洪. 干扰MAD2L1基因表达对乳腺癌细胞凋亡的影响及机制[J]. 山东大学学报 (医学版), 2022, 60(10): 9-16.

FENG Haigang, LIU Guowen, CAO Hong. Effects and mechanism of interfering MAD2L1 gene expression on the apoptosis of breast cancer cells[J]. Journal of Shandong University (Health Sciences), 2022, 60(10): 9-16.

参考文献

[1] Cerezo MV, Soria-Reyes LM, Alarcón R, et al. The satisfaction with life scale in breast cancer patients: psychometric properties [J]. Int J Clin Health Psychol, 2022, 22(1): 100274.
[2] Wu HJ, Chu PY. Recent discoveries of macromolecule- and cell-based biomarkers and therapeutic implications in breast cancer [J]. Int J Mol Sci, 2021, 22(2): 636.
[3] Wang X, Gao C, Feng F, et al. Construction and analysis of competing endogenous RNA networks for breast cancer based on TCGA dataset [J]. Biomed Res Int, 2020, 2020: 4078596. doi: 10.1155/2020/4078596.
[4] Zhu XF, Yi M, He J, et al. Pathological significance of MAD2L1 in breast cancer: an immunohistochemical study and meta analysis [J]. Int J Clin Exp Pathol, 2017, 10(9): 9190-9201.
[5] Ding X, Duan H, Luo H. Identification of core gene expression signature and key pathways in colorectal cancer [J]. Front Genet, 2020, 11: 45. doi: 10.3389/fgene.2020.00045.
[6] Wang Y, Wang F, He J, et al. miR-30a-3p targets MAD2L1 and regulates proliferation of gastric cancer cells [J]. Onco Targets Ther, 2019, 12: 11313-11324. doi: 10.2147/OTT.S222854.
[7] Li J, He X, Wu X, et al. miR-139-5p inhibits lung adenocarcinoma cell proliferation, migration, and invasion by targeting MAD2L1 [J]. Comput Math Methods Med, 2020, 2020: 2953598. doi: 10.1155/2020/2953598.
[8] Li Y, Bai W, Zhang J. MiR-200c-5p suppresses proliferation and metastasis of human hepatocellular carcinoma(HCC)via suppressing MAD2L1 [J]. Biomed Pharmacother, 2017, 92:1038-1044. doi: 10.1016/j.biopha.2017.05.092.
[9] Wu X, Peng L, Zhang Y, et al. Identification of key genes and pathways in cervical cancer by bioinformatics analysis [J]. Int J Med Sci, 2019, 16(6): 800-812.
[10] Su L, Zhang J, Zhang X, et al. Identification of cell cycle as the critical pathway modulated by exosome-derived microRNAs in gallbladder carcinoma [J]. Med Oncol, 2021, 38(12): 141.
[11] Gao Y, Liu Y, Sun L, et al. MAD2L1 functions as a novel diagnostic and predictive biomarker in cholangiocarcinoma[J]. Genet Test Mol Biomarkers, 2021, 25(11): 685-695.
[12] Xia T, Meng L, Zhao Z, et al. Bioinformatics prediction and experimental verification identify MAD2L1 and CCNB2 as diagnostic biomarkers of rhabdomyosarcoma [J]. Cancer Cell Int, 2021, 21(1): 634.
[13] Wu J, Lv Q, Huang H, et al. Screening and identification of key biomarkers in inflammatory breast cancer through integrated bioinformatic analyses [J]. Genet Test Mol Biomarkers, 2020, 24(8): 484-491.
[14] Ben-Hamo R, Jacob Berger A, Gavert N, et al. Predicting and affecting response to cancer therapy based on pathway-level biomarkers [J]. Nat Commun, 2020, 11(1): 3296.
[15] Wang Z, Katsaros D, Shen Y, et al. Biological and clinical significance of MAD2L1 and BUB1, genes frequently appearing in expression signatures for breast cancer prognosis [J]. PLoS One, 2015, 10(8): e0136246.
[16] Liu X, Chen Y, Li Y, et al. Targeting mitosis exit: a brake for cancer cell proliferation[J]. BiochimBiophys Acta Rev Cancer, 2019, 1871(1): 179-191.
[17] Ding X, Fu Q, Chen W, et al. Targeting of MAD2L1 by miR-515-5p involves the regulation of cell cycle arrest and apoptosis of colorectal cancer cells [J]. Cell Biol Int, 2022, 46(5): 840-848.
[18] Qi L, Zhou B, Chen J, et al. Significant prognostic values of differentially expressed-aberrantly methylated hub genes in breast cancer [J]. J Cancer, 2019, 10(26): 6618-6634.
[19] Young ND, Harris J, Evangelista M, et al. Diversity in the intrinsic apoptosis pathway of nematodes [J]. Commun Biol, 2020, 3(1): 49-63.
[20] Roche O, Fernández-Aroca DM, Arconada-Luque E, et al. p38β and cancer: the beginning of the road [J]. Int J Mol Sci, 2020, 21(20): 7524.
[21] 万强, 杨玉萍, 刘中勇. 丹参酮ⅡA通过抑制p38MAPK通路减轻PM2.5对血管内皮细胞的损伤[J].中国病理生理杂志, 2016, 32(4): 597-601. WAN Qiang, YANG Yuping, LIU Zhongyong. Tanshinone IIA attenuates PM2.5-induced vascular endothelial cell injury via p38 MAPK signal pathway [J]. Chinese Journal of Pathophysiology, 2016, 32(4): 597-601.
[22] 罗慧臣,胡丹慧,张济.miR-203-3p靶向TREM1基因调控TGF-β1/p38MAPK信号通路对狼疮性肾炎小鼠肾小管上皮细胞增殖和凋亡的影响[J].山东大学学报(医学版), 2021, 59(3): 18-25. LUO Huichen, HU Danhui, ZHANG Ji. Effect of miR-203-3p targeted TREM1 gene on the regulation of TGF-β1/p38MAPK signaling pathway on the proliferation and apoptosisof renal tubular epithelial cells in lupus nephritis mice [J]. Journal of Shandong University(Health Sciences), 2021, 59(3): 18-25.
[23] 朱金艳, 叶静怡, 刘志龙,等. 重楼单体PP-11对人乳腺癌MDA-MB-231细胞增殖的抑制作用及其机制[J].中国病理生理杂志, 2021, 37(2): 246-254. ZHU Jinyan,YE Jingyi, LIU Zhilong, et al. Inhibitory effect of PP-11, a monomer isolated from Paris polyphylla, onproliferation of human breast cancer MDA-MB-231 cells and its mechanism[J]. Chinese Journal of Pathophysiology, 2021, 37(2): 246-254.

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