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Changes of MicroRNA Expression and Apoptosis in Endometrial Glandular Epithelial Cells under Hypoxic
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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。 Abstract:Objective To explore the changes in the transcription levels of microRNAs(miRNAs) in endometrial glandular epithelial cells (EECs) under hypoxia and their effects on apoptosis. Methods EECs were seeded into six-well plates in logarithmic growth phase(1×105 cells/well), and divided into two groups: hypoxia group and control group. The cells in both hypoxia group and the control group were placed in a hypoxic environment (the volume ratio of O2∶N2∶CO2 was 1∶94∶5) and cultured in normoxic environment (O2∶CO2 volume ratio of 95∶5). All cells were collected after they were cultured 4 h, and Trizol was added into the cells and total RNAs were extracted. High-throughput sequencing was used to detect the changes of miRNAs expression profiles in the two groups of EECs. Subsequently, realtime fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the gene expression of miR-7704 and miR-7974. Flow cytometry was used to detect the apoptosis of EECs. The protein expression changes of p53 and apoptosis-related proteins were detected by Western blot. Results High-throughput sequencing detected the expression levels of 21 common miRNAs. Compared with the control group, 16 miRNAs were up-regulated and 5 miRNAs were down-regulated in the EECs of the hypoxia group; the expression of miR-7704 and miR-7974 decreased most significantly in the hypoxia group(all P < 0.05). RT-PCR results showed that compared with the control group, the relative expression levels of miR-7704 and miR-7974 in EECs of the hypoxia group decreased by 20% and 80%, respectively. The results of flow cytometry showed that the ratio of early apoptotic cells and late apoptotic cells in the hypoxia group was higher than that in the control group (all P < 0.001). The results of Western blot showed that the expression of p53 in EECs in the hypoxia group increased, and the expression of the anti-apoptotic protein B-cell lymphoma-2 decreased compared with the control group(all P < 0.05). Conclusions Hypoxic environment can induce changes in the expression profile of miRNAs in EECs, among which the down-regulation of miR-7974 is the most significant. p53 may be the target protein of miR-7974, and hypoxia-induced EEC apoptosis may be achieved by down-regulating the level of miR-7974 and promoting the expression of p53. -
Key words:
- endometrial glandular epithelial cells /
- hypoxic /
- miR-7704 /
- miR-7974 /
- apoptosis
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 -
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 
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[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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