DNA甲基化调控对男性不育的影响及其作用机制

Effect of DNA methylation regulation on male infertility and its mechanism

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。

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.