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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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