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中华临床医师杂志(电子版) ›› 2021, Vol. 15 ›› Issue (01) : 10 -16. doi: 10.3877/cma.j.issn.1674-0785.2021.01.002

所属专题： 文献；

临床研究

DNA甲基化调控对男性不育的影响及其作用机制
杜鹏1, 张晓丽2,†(), 赵晓勇2,†()   
  1. 1. 510010　广州，广东省妇幼保健院生殖健康与不孕症科
    2. 510800　广州，南方医科大学附属花都医院围产医学中心
  • 收稿日期:2020-09-02 出版日期:2021-01-15
  • 通信作者: 张晓丽, 赵晓勇
  • 基金资助:
    广东省自然科学基金(2016A030313419); 广东省医学科研基金(A2018328，B2019099); 广东省中医药局科技项目(20191256)

Effect of DNA methylation regulation on male infertility and its mechanism

Peng Du1, Xiaoli Zhang2,†(), Xiaoyong Zhao2,†()   

  1. 1. Department of Reproductive Health and Infertility, Guangdong Maternal and Child Health Hospital, Guangzhou 510010, China
    2. Center of Perinatal Medicine, Huadu Hospital Affiliated to Southern Medical University, Guangzhou 510800, China
  • Received:2020-09-02 Published:2021-01-15
  • Corresponding author: Xiaoli Zhang, Xiaoyong Zhao
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引用本文：

杜鹏, 张晓丽, 赵晓勇. DNA甲基化调控对男性不育的影响及其作用机制[J]. 中华临床医师杂志(电子版), 2021, 15(01): 10-16.

Peng Du, Xiaoli Zhang, Xiaoyong Zhao. Effect of DNA methylation regulation on male infertility and its mechanism[J]. Chinese Journal of Clinicians(Electronic Edition), 2021, 15(01): 10-16.

目的

探讨表观遗传学调控对男性不育的影响及作用机制。

方法

选择2020年2月至12月于广东省妇幼保健院生殖健康与不孕症科门诊就诊患者作为研究对象，分为对照组（50例）及不育组（50例）。收集精液标本，采用Real time-PCR和Western blot检测DNMT1、ERp29、PTEN、TSC2 mRNA和蛋白表达，采用甲基化DNA免疫共沉淀测序（MeDIP-Seq）检测甲基化差异显著的基因。采用GC1、GC2、TM4精子细胞株，分为空白对照组（Con组）、ERp29沉默组（shERp29组）、ERp29过表达组（ERp29OE组）。去甲基药物（5-AZA-DC）处理后进行甲基化特异性PCR（MSP）、细胞增殖、凋亡能力检测。

结果

与对照组相比，不育组患者DNMT1 mRNA［5.25±1.12 vs 8.22±0.67，P＜0.05］和蛋白［3.33±0.98 vs 7.31±0.55，P＜0.01］表达上调，差异具有统计学意义；ERp29 mRNA［5.17±0.98 vs 2.27±0.33，P＜0.05］和蛋白［6.12±1.03 vs 2.59±0.43，P＜0.05］、PTEN mRNA［4.58±0.77 vs 1.97±0.28，P＜0.05］和蛋白［6.21±1.04 vs 2.28±0.36，P＜0.05］、TSC2 mRNA［6.39±0.83 vs 3.16±0.52，P＜0.05］和蛋白［6.56±0.67 vs 3.03±0.17，P＜0.05］表达下调，差异具有统计学意义。MeDIP-Seq结果显示，不育组中ERp29、PTEN和TSC2启动子区域甲基化水平差异显著，有统计学意义（P＜0.05）。MSP证实GC1细胞株PTEN基因启动子区CpG岛DNA甲基化，5-AZA-DC干预前shERp29、ERp29OE 组甲基化上升，干预后shERp29、ERp29OE 组甲基化下降。GC1、GC2、TM4细胞株5-AZA-DC干预后shERp29和ERp29OE组增殖上调，凋亡下降，差异有统计学意义（P＜0.05），Con组无统计学意义（P＞0.05）。

结论

ERp29是引起男性不育发病的重要基因，可作为男性不育表观遗传学治疗的潜在靶点。

关键词: 表观遗传学, DNA甲基化, 男性不育
Objective

To investigate the effect of epigenetic regulation on male infertility and its underlying mechanism.

Methods

From February to December 2020, outpatients at Department of Reproductive Health and Infertility of Guangdong Maternal and Child Health Hospital were selected and divided into either a control group (n=50) or a sterility group (n=50). Semen samples were collected for real-time PCR and Western blot detection of DNMT1, ERp29, PTEN, and TSC2 mRNA and protein. DNA methylation immunoprecipitation sequencing (MeDIP-seq) was used to detect genes with significant methylation differences. GC1, GC2, and TM4 sperm cell lines were divided into a blank control group (con group), ERp29 silencing group (shERp29 group), and ERp29 overexpression group (ERp29OEgroup). The demethylating drug 5-AZA-DC was used to treat cells, and methylation-specific PCR (MSP), CCK-8, and flow cytometry were then performed to detect cell proliferation and apoptosis.

Results

Compared with the control group, DNMT1 mRNA (5.25±1.12 vs 8.22±0.67, P＜0.05) and protein (3.33±0.98 vs 7.31±0.55, P＜0.05) expression was up-regulated, while ERp29 mRNA ([5.17±0.98 vs 2.27±0.33, P＜0.05) and protein (6.12±1.03 vs 2.59±0.43, P＜0.05), PTEN mRNA (4.58±0.77 vs 1.97±0.28, P＜0.05) and protein (6.21±1.04 vs 2.28±0.36, P＜0.05), and TSC2 mRNA (6.39±0.83 vs 3.16±0.52, P＜0.05) and protein (6.56±0.67 vs 3.03±0.17, P＜0.05) expression were down-regulated. The MeDIP-Seq results showed significant differences in methylation levels of ERp29, PTEN, and TSC2 promoter regions in the sterility group (P＜0.05). MSP confirmed the DNA methylation of CPG islands in the promoter region of PTEN gene in the GC1 cell line. Before 5-AZA-DC intervention, the shERp29 and ERp29OE group had increased methylation, and after intervention, the two groups had decreased methylation. After intervention of GC1, GC2, and TM4 cell lines with 5-AZA-DC, cell proliferation in the shERp29 and ERp29OE groups was significantly enhanced and apoptosis significantly decreased (P＜0.05), although there was no significant difference in the con group (P＞0.05).

Conclusion

ERp29 is an important gene that causes male infertility and can be a potential target for epigenetic treatment of male infertility.

Key words: Epigenetics, DNA methylation, Male infertility
表1 2组男性精液中DNMT1、ERp29、PTEN、TSC2 mRNA表达情况（
xˉ
±s）
组别 例数 DNMT1 ERp29 PTEN TSC2
对照组 50 5.25±1.12 5.17±0.98 4.58±0.77 6.39±0.83
不育组 50 8.22±0.67 2.27±0.33 1.97±0.28 3.16±0.52
t值 2.987 2.881 2.863 3.015
P值 0.019 0.023 0.025 0.016
表2 2组男性精液中DNMT1、ERP29、PTEN、TSC2蛋白表达情况（
xˉ
±s）
组别 例数 DNMT1 ERp29 PTEN TSC2
对照组 50 3.33±0.98 6.12±1.03 6.21±1.04 6.56±0.67
不育组 50 7.31±0.55 2.59±0.43 2.28±0.36 3.03±0.17
t值 2.774 2.778 2.854 2.882
P值 0.027 0.026 0.025 0.023
图1 2组精液差异显著的基因启动子区域DNA甲基化情况。图a为2组精液差异显著基因启动子区域DNA甲基化聚类图；图b为MeDIP-Seq分析精液ERp29、PTEN、TSC2甲基化差异情况
表3 2组男性精液ERp29、PTEN、TSC2启动子区域甲基化水平（
xˉ
±s）
组别 例数 ERp29 PTEN TSC2
对照组 50 10.53±1.21 9.38±1.04 9.53±1.49
不育组 50 17.69±1.88 13.67±1.12 13.84±1.51
t值 2.883 2.815 2.941
P值 0.023 0.026 0.021
图2 5-AZA-DC干预前后GC1细胞株PTEN启动子甲基化情况
图3 CCK8检测各组5-AZA-DC干预前后细胞增殖情况（n=3）。图a为GC1细胞;图b为GC2细胞;图c为TM4细胞
图4 流式细胞仪检测5-AZA-DC干预前后3种细胞凋亡情况（n=3）。图a为GC1细胞;图b为GC2细胞;图c为TM4细胞
表4 5-AZA-DC干预前后3组细胞凋亡率比较（
xˉ
±s，n=3）
组别 5-AZA-DC（-） 5-AZA-DC（+）
GC1 GC2 TM4 GC1 GC2 TM4
Con组 7.21±1.03 7.23±1.02 7.20±1.01 7.22±1.01 7.20±1.03 7.22±1.02
shERp29组 17.24±1.53 15.26±1.68 15.33±1.49 10.25±1.35* 13.34±1.28* 13.32±1.29*
ERp29OE组 15.29±1.52 13.24±1.31 13.26±1.32 8.26±1.05* 9.37±1.14* 9.38±1.15*
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