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缺氧环境下子宫内膜腺上皮细胞中微RNA表达谱及细胞凋亡变化

王含必 窦帅杰 刘思邈 张婉玉 刘美芝 邓成艳

王含必, 窦帅杰, 刘思邈, 张婉玉, 刘美芝, 邓成艳. 缺氧环境下子宫内膜腺上皮细胞中微RNA表达谱及细胞凋亡变化[J]. 协和医学杂志, 2022, 13(4): 626-631. doi: 10.12290/xhyxzz.2022-0095
引用本文: 王含必, 窦帅杰, 刘思邈, 张婉玉, 刘美芝, 邓成艳. 缺氧环境下子宫内膜腺上皮细胞中微RNA表达谱及细胞凋亡变化[J]. 协和医学杂志, 2022, 13(4): 626-631. doi: 10.12290/xhyxzz.2022-0095
WANG Hanbi, DOU Shuaijie, LIU Simiao, ZHANG Wanyu, LIU Meizhi, DENG Chengyan. Changes of MicroRNA Expression and Apoptosis in Endometrial Glandular Epithelial Cells under Hypoxic[J]. Medical Journal of Peking Union Medical College Hospital, 2022, 13(4): 626-631. doi: 10.12290/xhyxzz.2022-0095
Citation: WANG Hanbi, DOU Shuaijie, LIU Simiao, ZHANG Wanyu, LIU Meizhi, DENG Chengyan. Changes of MicroRNA Expression and Apoptosis in Endometrial Glandular Epithelial Cells under Hypoxic[J]. Medical Journal of Peking Union Medical College Hospital, 2022, 13(4): 626-631. doi: 10.12290/xhyxzz.2022-0095

缺氧环境下子宫内膜腺上皮细胞中微RNA表达谱及细胞凋亡变化

doi: 10.12290/xhyxzz.2022-0095
基金项目: 

中华医学会临床医学科研专项基金“干细胞基础研究”项目 19020020781

中国医学科学院中央级公益性科研院所基本科研业务费专项基金 2021-PT320-001

详细信息
    通讯作者:

    邓成艳, E-mail: chydmd@sohu.com

    王含必、窦帅杰对本文同等贡献

    王含必、窦帅杰对本文同等贡献

  • 中图分类号: R711

Changes of MicroRNA Expression and Apoptosis in Endometrial Glandular Epithelial Cells under Hypoxic

Funds: 

Chinese Medical Association Clinical Medical Research Fund 19020020781

Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences 2021-PT320-001

More Information
  • 摘要:   目的  探究缺氧环境下子宫内膜腺上皮细胞(endometrial glandular epithelial cell, EEC)中微RNA(micro-RNA,miRNA)表达谱变化及其对凋亡的影响。  方法  取对数生长期EEC,接种至六孔板(1×105个细胞/孔)并随机分为缺氧组和对照组。缺氧组、对照组分别置于缺氧环境(O2∶N2∶CO2体积比为1∶94∶5)和正常氧环境(O2∶CO2体积比为95∶5)中培养。培养4 h后收集细胞,加入Trizol并提取总RNA。采用高通量测序法测定两组EEC中miRNA表达谱变化,采用实时荧光定量PCR(realtime fluorescence quantitative polymerase chain reaction, RT-PCR)法检测miR-7704、miR-7974基因表达水平,采用流式细胞术检测EEC凋亡情况,采用蛋白印迹法(Western blot)检测p53和凋亡相关蛋白表达变化。  结果  高通量测序对21种常见miRNA表达水平检测后发现,与对照组比较,缺氧组EEC中16种miRNA表达上调,5种miRNA表达下调;其中对照组表达丰度较高的miR-7704和miR-7974在缺氧组表达下降最为显著(P均<0.05)。RT-PCR结果显示,与对照组比较,缺氧组EEC中miR-7704和miR-7974相对表达量分别降低20%、80%。流式细胞术检测结果显示,缺氧组早期凋亡率及晚期凋亡率均高于对照组(P均<0.001)。Western blot检测结果显示,与对照组比较,缺氧组EEC中p53表达升高,抗凋亡蛋白B细胞淋巴瘤-2表达降低(P均<0.05)。  结论  缺氧环境可诱导EEC中miRNA表达谱改变,其中以miR-7974表达下调最为显著。p53可能是miR-7974的靶蛋白,缺氧诱发的EEC凋亡可能通过下调miR-7974水平而促进p53表达实现。
    作者贡献:王含必负责研究设计、数据分析、论文撰写;窦帅杰负责实验实施、数据分析、论文撰写;刘思邈、张婉玉、刘美芝负责实验实施;邓成艳为项目总负责人,指导研究设计、论文写作及修改。
    利益冲突:所有作者均声明不存在利益冲突
  • 图  1  p53是miR-7974潜在的靶基因

    A.生物信息学网站预测miR-7974的靶基因为p53;B. miR-7974与p53的3'非翻译区相结合

    图  2  缺氧组和对照组EEC中微RNA表达谱的聚类热图

    EEC:子宫内膜腺上皮细胞

    图  3  缺氧组和对照组EEC中miR-7704和miR-7974相对表达量的柱状图

    EEC:同图 2;与对照组比较,*P<0.05,* * P<0.001

    图  4  缺氧组和对照组EEC凋亡的流式细胞图

    EEC:同图 2]]> 缺氧环境诱导miR-7974的靶蛋白p53表达上升 Western blot检测结果显示,与对照组比较,缺氧组EEC中p53蛋白表达升高,伴抗凋亡蛋白Bcl-2表达降低(P均<0.05),Cleaved-caspase3无显著变化(P>0.05),提示缺氧诱发EEC中miR-7974表达下调,可能通过上调其靶蛋白p53水平进而诱导细胞凋亡(图 5)。 缺氧组和对照组凝胶电泳图 Bcl-2:B细胞淋巴瘤-2

    表  1  RT-PCR引物序列

    引物 序列(5′→3′)
    snoRNA202 上游引物:ACACTCCAGCTGGGGCTGTACTGACTTGATG
    下游引物:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCATCAGAT
    URP-R TGGTGTCGTGGAGTCG
    miR-7974 上游引物:ACACTCCAGCTGGGAGGCTGTGATGCTCCCT
    下游引物:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGCTCAG
    miR-7704 上游引物:ACACTCCAGCTGGGCGGGGTCGGCGGC
    下游引物:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACGTCGC
    RT-PCR:实时荧光定量聚合酶链式反应
    下载: 导出CSV
  • [1] Casper RF, Yanushpolsky EH. Optimal endometrial preparation for frozen embryo transfer cycles: window of implanta-tion and progesterone support[J]. Fertil Steril, 2016, 105: 867-872. doi:  10.1016/j.fertnstert.2016.01.006
    [2] Altmäe S, Koel M, Võsa U, et al. Meta-signature of human endometrial receptivity: a meta-analysis and validation study of transcriptomic biomarkers[J]. Sci Rep, 2017, 30, 7: 10077. https://www.nature.com/articles/s41598-017-10098-3
    [3] Ranisavljevic N, Raad J, Anahory T, et al. Embryo transfer strategy and therapeutic options in infertile patients with thin endometrium: a systematic review[J]. J Assist Reprod Genet, 2019, 36: 2217-2231. doi:  10.1007/s10815-019-01576-w
    [4] 杨晓清, 张沐. 子宫内膜间质细胞体外损伤模型的建立[J]. 中华妇产科杂志, 2012, 47: 274-280. doi:  10.3760/cma.j.issn.0529-567x.2012.04.008
    [5] Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF[J]. Hum Reprod, 2000, 15: 806-809. doi:  10.1093/humrep/15.4.806
    [6] Zhang D, Han M, Zhou M, et al. Down-regulation of S100P induces apoptosis in endometrial epithelial cell during GnRH antagonist protocol[J]. Reprod Biol Endocrinol, 2021, 19: 99. doi:  10.1186/s12958-021-00787-0
    [7] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116: 281-297. doi:  10.1016/S0092-8674(04)00045-5
    [8] Mohr AM, Mott JL. Overview of microRNA biology[J]. Semin Liver Dis, 2015, 35: 3-11. doi:  10.1055/s-0034-1397344
    [9] Bister N, Pistono C, Huremagic B, et al. Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions[J]. J Extracell Vesicles, 2020, 10: e12002.
    [10] Semenza GL. Perspectives on oxygen sensing[J]. Cell, 1999, 98: 281-284. doi:  10.1016/S0092-8674(00)81957-1
    [11] Zheng J, Dai Y, Lin X, et al. Hypoxiainduced lactate dehydrogenase A protects cells from apoptosis in endometriosis[J]. Mol Med Rep, 2021, 24: 637. doi:  10.3892/mmr.2021.12276
    [12] Wawryk-Gawda E, Chylinska-Wrzos P, Lis-Sochocka M, et al. P53 protein in proliferation, repair and apoptosis of cells[J]. Protoplasma, 2014, 251: 525-533. doi:  10.1007/s00709-013-0548-1
    [13] Changizi V, Azadbakht O, Ghanavati R, et al. Effect of Lactobacillus species on apoptosis-related genes BCL2, BAX, and caspase 3 in the testes of gamma-irradiated rats[J]. Rev Assoc Med Bras, 2021, 67: 1581-1585. doi:  10.1590/1806-9282.20210634
    [14] Chen PS, Chiu WT, Hsu PL, et al. Pathophysiological implications of hypoxia in human diseases[J]. J Biomed Sci, 2020, 27: 63. doi:  10.1186/s12929-020-00658-7
    [15] Li Y, Lv X, Jiang M, et al. Sitagliptin ameliorates hypoxia-induced damages in endometrial stromal cells: an implication in endometriosis[J]. Bioengineered, 2022, 13: 800-809. doi:  10.1080/21655979.2021.2012950
    [16] Laptenko O, Prives C. Transcriptional regulation by p53: one protein, many possibilities[J]. Cell Death Differ, 2006, 13: 951-961. doi:  10.1038/sj.cdd.4401916
    [17] Luanpitpong S, Chanvorachote P, Stehlik C, et al. Regulation of apoptosis by Bcl-2 cysteine oxidation in human lung epithelial cells[J]. Mol Biol Cell, 2013, 24: 858-869. doi:  10.1091/mbc.e12-10-0747
    [18] Cho SO, Lim JW, Kim H. Diphenyleneiodonium inhibits apoptotic cell death of gastric epithelial cells infected with helicobacter pylori in a Korean isolate[J]. Yonsei Med J, 2015, 56: 1150-1154. doi:  10.3349/ymj.2015.56.4.1150
    [19] Li J, Yanyan M, Mu L, et al. The expression of Bcl-2 in adenomyosis and its effect on proliferation, migration, and apoptosis of endometrial stromal cells[J]. Pathol Res Pract, 2019, 215: 152477. doi:  10.1016/j.prp.2019.152477
    [20] Suroto H, Asriel A, De Vega B, et al. Early and late apoptosis protein expression (Bcl-2, BAX and p53) in traumatic brachial plexus injury[J]. J Musculoskelet Neuronal Interact, 2021, 21: 528-532.
    [21] Li YN, Ning N, Song L, et al. Derivatives of deoxypodophyllotoxin induce apoptosis through Bcl-2/Bax proteins expression[J]. Anticancer Agents Med Chem, 2021, 21: 611-620. doi:  10.2174/1871520620999200730160952
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出版历程
  • 收稿日期:  2022-03-02
  • 录用日期:  2022-04-13
  • 刊出日期:  2022-07-30

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