DNA甲基化参与调控Dazl生殖特异表达及其与男性生精障碍和肿瘤的相关性研究

发布时间：2018-07-04 18:40

  本文选题：Dazl基因 + DNA甲基化　； 参考：《南京医科大学》2015年博士论文

【摘要】：DAZL是高度保守的DAZ(Deleted in Azoospermia)家族成员之一,定位于脊椎动物的常染色体,在PGC发育迁移、精原细胞分化、减数分裂起始及发展等过程中起重要作用,人DAZL蛋白可以促进胚胎干细胞分化为精子,是配子生成的重要调控因子。DAZL同源基因在从硬骨鱼到人类几乎所有物种的生殖细胞中特定表达,其表达持续存在于胚胎干细胞(ESCs)直至单倍体圆形精子。是什么机制调控了Daz1高度时空特异性表达?这是我们面临的科学问题,DNA甲基化在内的表观遗传修饰是可能的表达调控机制。精子形成过程中,存在原始生殖细胞甲基化信息广泛擦除和随后生殖细胞甲基化的重新建立,这对保障精子的正常发育起重要作用,异常的表观遗传修饰尤其是DNA甲基化可能是导致男性生精障碍及精子活力下降的机制之一。生精相关基因Daz1启动子甲基化模式异常改变是否与男性生精障碍存在关联?部分生殖特异表达基因在人类体细胞恶性肿瘤中异常激活称为癌-生殖基因,研究生殖特定基因Daz1的表达调控机制以及鉴定癌-生殖基因能够为恶性肿瘤相关基础研究提供帮助。本研究分二部分：一、DNA甲基化参与调控Daz1生殖特异表达及其在物种间进化上的保守性该部分研究首先检测了Daz1在小鼠睾丸和体细胞组织中的表达差异,进而测定Daz1启动子CpG岛在不同组织类型中的甲基化模式,分析小鼠Daz1启动子甲基化模式与Daz1特定表达的关系；进一步研究了解小鼠精子发生不同阶段中Daz1表达是否和其甲基化模式存在关联；在体细胞系(NIH3T3)中采用5-氮杂-2’-脱氧胞苷处理去甲基化后检测Daz1表达情况；在其他哺乳动物和脊椎动物中选取不同物种进行研究,了解Daz1转录调控机制在进化上的保守性;设定计算法对Daz1启动子区域进行生物信息学分析,预测可能的转录因子结合位点。通过以上实验力图阐明Daz1生殖特异表达的转录调控机制。成果如下：1.小鼠Daz1基因表达存在时空特异性通过qRT-PCR方法检测Daz1在成年雄性小鼠睾丸、肝脏、心脏、肠管、脾脏、肌肉、肾脏、胸腺、肺及脑组织中的表达。在体细胞组织中没有检测到Daz1mRNA的表达,而在小鼠睾丸中高表达。继续用qRT-PCR方法检测Daz1在生后3天、6天、8天、12天、14天、16天、18天、20天、21天、5周小鼠睾丸中的表达。在不同发育阶段的小鼠睾丸中,Daz1在3日龄小鼠睾丸中检测出表达,于出生后16-18天时达到峰值,其后有所降低。实验室前期进行的小鼠睾丸冰冻切片免疫组化实验表明Daz1在精原细胞、前细线期精母细胞、偶线期精母细胞和早-中粗线期精母细胞中高表达,此后表达渐减少,在圆形精子中有少量表达,于长形精子中未检测到。通过以上研究,表明Daz1基因表达模式具有高度时空特异性。2.小鼠Daz1启动子CpG岛的甲基化模式与基因表达呈现显著负相关我们运用Methyl Primer Expresss V1.0软件对Daz1启动子进行分析,预测出相应区域CpG岛,采用亚硫酸氢盐修饰后测序PCR(Bisulfite sequencing PCR, BSP)法,分别检测不同组织细胞类型Daz1启动子CpG岛的甲基化状态。结果显示：在成年小鼠体细胞组织(肾脏、脑)中Daz1启动子CpG岛高度甲基化,而在睾丸中Daz1启动子CpG岛呈现低甲基化模式,附睾精子中甲基化程度相较睾丸组织更低。Daz1启动子CpG岛的甲基化模式与其表达情况呈现明显的负相关,提示Daz1启动子区域甲基化可能参与了Daz1生殖细胞特异性表达的调控。通过对小鼠出生后不同时间点睾丸中Daz1启动子CpG岛的甲基化检测,我们发现Daz1启动子区域在小鼠6-8日龄以及10-12日龄呈现低甲基化状态,而在出生后20-24天表现为甲基化程度升高,这一结果与小鼠Daz1表达于精原细胞、持续到圆形精子、以及后续阶段的表达降低相一致。以上相关性结果提示DNA甲基化参与调控了小鼠Daz1时间(精子发生阶段)空间(生殖细胞)特异性的表达。3.体细胞中进行去甲基化处理激活Daz1异常表达上述实验显示小鼠Daz1表达与其启动子甲基化状态呈现显著负相关关系,提示DNA甲基化参与调控小鼠Daz1的时空特异表达,那么在Daz1不表达的体细胞中采用药物去甲基化是否能够诱导Daz1异常激活?为此,我们采用甲基化抑制剂5-Aza-CdR在体细胞系进行去甲基化处理,通过qRT-PCR检测5-Aza-CdR处理后Daz1 mRNA的水平。结果显示Daz1在去甲基化处理后的体细胞中表达升高,作为对照的Tex19.1和RHOX2亦异常升高,而DAZ家族另一成员Boule却无明显变化。这一结果提示体细胞中Daz1的表达沉默由其基因启动子甲基化模式调控,而Boule表达调控机制可能更为复杂多元。4.DNA甲基化机制参与调控Daz1生殖特异表达在物种进化上存在高度保守性。实验采用qRT-PCR对收集的人睾丸及体细胞组织(结肠、前列腺、胃、胆囊及脾脏)DAZL mRNA表达水平进行检测,结果显示DAZL mRNA在人睾丸组织中高表达,而在体细胞组织中不表达。进一步研究DAZL启动子甲基化模式是否与此相关,实验提取人睾丸、精子、体细胞组织(前列腺、脾脏)DNA进行甲基化检测,发现在体细胞组织中DAZL启动子为高甲基化状态而在睾丸中显示为低甲基化,精子中显示为更低的甲基化程度。相似的实验结果在另一种哺乳动物猪中同样存在,表明DNA甲基化机制参与调控Daz1生殖特异表达存在于哺乳动物中。鉴于Daz1最早表达于脊椎动物生殖细胞,我们进一步实验了解这一调控机制在进化上是否在更低级别的脊椎动物中存在。我们提取了斑马鱼和莱航鸡性腺及体细胞组织的RNA及基因组DNA,实验结果显示Daz1 mRNA在性腺中高表达而Daz1启动子呈低甲基化状态,体细胞组织中Daz1启动子呈高甲基化而无Daz1 mRNA表达。Daz1同源基因表达从硬骨鱼类开始出现到哺乳动物直至人类,以上实验表明启动子区差异化的甲基化模式参与调控Daz1生殖特异性表达,支持这一调控机制古老并且在物种进化上维持保守性的假说。5. Daz1启动子区转录因子可能结合位点的生物信息学预测分析预测转录因子结合位点是研究基因转录调控的重要组成部分。前述研究明确了Daz1在生殖细胞和体细胞中差异化的甲基化模式和其生殖特异表达密切相关,且这种关联贯穿Daz1整个基因进化历程,表明DNA甲基化调控机制在Daz1基因表达调控中保守存在。那么,Dazl启动子区域是否存在保守的顺式作用元件参与基因的转录调控?我们检索人、小鼠、猪及大鼠Dazl Exonl上游3.5kb的序列,设定计算法检测长度大于7个碱基的保守序列,结果显示在上述物种中共同存在12个由7个核苷酸组成的相同序列,数据库检索发现这些保守存在的相似序列可能是一些转录因子的结合位点。二、DAZL表达调控异常与男性生精障碍及肿瘤相关性研究。1.DAZL启动子异常的甲基化模式与男性生精障碍有关。精子发生过程经历了基因组范围内的去甲基化和重新甲基化过程,男性精子甲基化的研究工作将从表观遗传学角度为男性生精障碍临床诊疗提供帮助。为此,我们比较了正常男性与少弱精子症患者精子DAZL启动子甲基化谱差异。收集精液标本后进行精液常规检查和精子形态测定；采用密度梯度离心法去除体细胞污染,提取精子基因组DNA；亚硫酸氢盐处理后PCR体外扩增并纯化,与pCR2.1载体连接及酶切验证,挑取阳性克隆测序；分析不同样本精子DNA甲基化程度差异。和对照相比,少弱精症患者DAZL启动子甲基化率明显升高,结果存在统计学差异。这一结果提示DAZL启动子区异常的甲基化模式可能与男性生精障碍有关。2.DAZL在部分恶性肿瘤中异常表达。癌-生殖基因或癌-睾丸基因是指一类其抗原蛋白表达于人类多种组织来源恶性肿瘤,而在正常组织中只存在于睾丸、卵巢或胎盘组织。上研究显示Dazl生殖细胞特定表达与其基因调控区甲基化模式差异密切相关,其是否类似于癌-生殖基因在恶性肿瘤中异常表达?我们在TCGA数据库中搜索发现DAZL在头颈部肿瘤、肺癌和乳腺癌中表达,尤其在后者中表达较明显。但和其他经典癌-生殖基因相比,DAZL在肿瘤中表达量仍较低,提示DAZL在部分肿瘤中异常表达可能和肿瘤细胞基因组整体异常低甲基化有关。
[Abstract]:DAZL is one of the highly conserved members of the DAZ (Deleted in Azoospermia) family. It is located in the autosomes of vertebrates. It plays an important role in the process of PGC development and migration, spermatogonial differentiation, meiosis initiation and development. Human DAZL protein can promote the differentiation of embryonic stem cells into sperm, which is an important regulatory factor of gamete generation,.DAZL. The source genes are expressed in the germ cells of almost all species from the hard bone fish to the human species, and their expression persists in the embryonic stem cells (ESCs) until the haploid round sperm. What mechanism regulates the high temporal and spatial specific expression of Daz1? This is the scientific problem we face, and the epigenetic modification, including DNA methylation, is a possible table. In the process of sperm formation, there is a widespread erasure of the methylation information of the primitive germ cells and the reestablishment of the subsequent methylation of the germ cells, which plays an important role in ensuring the normal development of sperm. Abnormal epigenetic modification, especially DNA methylation, may be the mechanism leading to male spermatogenesis disorder and the decline of sperm motility. 1. Is the abnormal change of the Daz1 promoter methylation pattern associated with male spermatogenesis disorders? The abnormal activation of partial reproductive specific genes in human body cell malignant tumors is called the cancer reproductive gene, studies the regulation mechanism of the expression of the specific reproductive gene Daz1 and the identification of the cancer reproductive genes for malignant tumors. The relevant basic research provides help. This study is divided into two parts: first, DNA methylation participates in the regulation of Daz1 reproductive specific expression and its conservatism in interspecies evolution. This part of the study first detected the difference in the expression of Daz1 in mouse testis and somatic cells, and then measured the methylation of the Daz1 promoter CpG island in different tissue types. Model analysis of the relationship between Daz1 promoter methylation and Daz1 specific expression in mice; further study whether Daz1 expression in different stages of spermatogenesis in mice was associated with its methylation pattern; in somatic cell line (NIH3T3), 5- n-heterozygous -2 'deoxycytidine demethylation was used to detect the expression of Daz1; The selection of different species in mammals and vertebrates is conducted to understand the evolutionary conservatism of the Daz1 transcriptional regulation mechanism; to set a computational method for the bioinformatics analysis of the Daz1 promoter region and to predict the possible transcription factor binding site. Through the above experiments, the transcriptional regulation mechanism of the specific expression of Daz1 is clarified. As follows: 1. the expression of Daz1 gene in mice was specific by qRT-PCR method to detect the expression of Daz1 in the testis, liver, heart, intestines, intestines, spleen, muscle, kidney, thymus, lung and brain tissue of adult male mice. The expression of Daz1mRNA was not detected in somatic tissue and high expression in mouse testis. QRT-PCR method continued to be used. The expression of Daz1 was detected in mice testis at 3 days, 6 days, 8 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days and 5 weeks. In mice testis at different developmental stages, Daz1 was detected in the testicles of 3 days of age in mice, and reached their peak value after birth and then decreased. The frozen section of testis in the early laboratory of mice was immunized. The results showed that Daz1 was highly expressed in spermatogonia, pre fine line spermatocyte, dipinogenic spermatocyte, and early middle roughen stage spermatocyte, and then decreased gradually. There was a small amount of expression in round sperm and not detected in long spermatozoa. Through the above study, the Daz1 gene expression pattern has a highly spatio-temporal specific.2. mouse Daz1 startup. The methylation pattern of the sub CpG island has a significant negative correlation with the gene expression. We use Methyl Primer Expresss V1.0 software to analyze Daz1 promoter, predict the corresponding region CpG Island, use the hydrogen sulfite modified sequence PCR (Bisulfite sequencing PCR, BSP) method, and separate the different tissue cell types to detect the promoter Island. Methylation status. The results showed that the Daz1 promoter CpG island was highly methylation in the adult mouse somatic tissue (kidney, brain), and the Daz1 promoter CpG island in the testis showed a low methylation pattern. The methylation level in the epididymal sperm was lower than that of the testicular tissue, and the methylation pattern of the.Daz1 promoter CpG island was obviously negative. Correlation, suggesting that methylation of the Daz1 promoter region may be involved in the regulation of the specific expression of Daz1 germ cells. By methylation of the Daz1 promoter CpG island in the testicles at different time points after birth, we found that the Daz1 promoter region showed low methylation status at 6-8 days and 10-12 days of age in mice, and 20-24 days after birth. The results showed that the degree of methylation was elevated, which was consistent with the expression of Daz1 in spermatogonial cells, round spermatozoa and subsequent stages. The above correlation results suggest that DNA methylation participates in the regulation of Daz1 time (spermatogenesis) space (spermatogenic cell) specific expression of.3. somatic cells in mice. The above experiments of activation of Daz1 showed that the expression of Daz1 in mice was negatively correlated with the methylation status of the promoter, suggesting that DNA methylation participates in the time and space specific expression of Daz1 in mice. Then, whether the use of drug demethylation in Daz1 non expressed somatic cells can induce abnormal activation of Daz1? For this reason, Methylation inhibitor 5-Aza-CdR was used to demethylation in somatic cell lines, and the level of Daz1 mRNA after 5-Aza-CdR treatment was detected by qRT-PCR. The results showed that the expression of Daz1 was increased in the somatic cells after demethylation treatment, and the control Tex19.1 and RHOX2 also increased, but the Boule of the other member of the DAZ family did not change significantly. The results suggest that the expression of Daz1 in somatic cells is regulated by the methylation mode of its gene promoter, and the regulatory mechanism of Boule expression may be more complex and multiple.4.DNA methylation mechanism involved in the regulation of Daz1 reproduction specific expression in the species evolution. The experimental qRT-PCR was used to collect human testis and somatic tissue (colon,) The expression level of DAZL mRNA in the prostate, stomach, gallbladder and spleen was detected. The results showed that DAZL mRNA was highly expressed in human testicular tissue, but was not expressed in somatic tissue. Further study of the correlation between the Zi Jiaji mode of DAZL initiation and the experimental extraction of DNA from human testis, spermatozoa and somatic cells (prostate, spleen) DNA was detected. It was found that the DAZL promoter was methylation in the somatic tissue and showed low methylation in the testis, and the sperm showed a lower degree of methylation. Similar experimental results were found in another mammal pig, indicating that the DNA methylation mechanism involved in the regulation of Daz1 reproductive specific expression in mammals. In view of Daz1 It was first expressed in vertebrate germ cells. We further studied whether this regulatory mechanism existed in lower levels of vertebrates. We extracted the RNA and genomic DNA of the gonads and somatic tissues of zebrafish and leihang chickens. The experimental results showed that the Daz1 mRNA was highly expressed in the gonads and the Daz1 promoter was low. In the basic state, the Daz1 promoter in somatic tissue is hypermethylation and no Daz1 mRNA expression.Daz1 homologous gene expression from the bone fishes to mammalian and human. The above experiments show that the differential methylation mode of the promoter region participates in the regulation of Daz1 reproductive specificity, and supports this regulatory mechanism in ancient and in species. Evolutionary conservatism hypothesis.5. Daz1 promoter region transcription factor potential binding site bioinformatics prediction analysis and prediction of transcription factor binding site is an important component of gene transcription regulation. The previous study clarified the differential methylation pattern and its reproductive specific expression in Daz1 in germ cells and somatic cells. Closely related, and this association runs through the entire genetic evolution of Daz1, indicating that the regulatory mechanism of DNA methylation exists conservatively in the regulation of Daz1 gene expression. Then, is there a conservative cis acting element in the Dazl promoter region to participate in gene transcription regulation? We retrieve the sequence of 3.5kb in the upstream of human, mice, pigs and rat Dazl Exonl. The results showed that there were 12 identical sequences of 7 nucleotides in the above species, and the database retrieval found that these conserved similar sequences might be the binding sites of some transcription factors. Two, the abnormal regulation of DAZL expression was associated with male spermatogenesis and cancer. The methylation pattern of the.1.DAZL promoter anomaly is related to the male spermatogenesis disorder. The process of spermatogenesis has undergone the process of demethylation and re methylation within the genome range. The study of male sperm methylation will help the clinical diagnosis and treatment of male spermatogenesis from the epigenetic point of view. Sperm DAZL promoter methylation spectrum difference between male and oligozoospermia patients. Semen specimens were collected for semen routine examination and sperm morphometry; density gradient centrifugation was used to remove somatic cell pollution and to extract sperm genome DNA; PCR was amplified and purified in vitro after hydrogen sulphite treatment, connected with pCR2.1 vector and verified by enzyme digestion. The positive clones were sequenced and the difference of DNA methylation in different samples of sperm was analyzed. Compared with the control, the methylation rate of DAZL promoter was significantly higher in the patients with oligoasthenospermia. The results suggested that the abnormal methylation pattern in the DAZL promoter region may be associated with the male spermatogenesis disorders associated with.2.DAZL in some malignant tumors. Abnormal expression. Cancer - reproductive gene or cancer - testicular gene is a class of antigen protein expressed in a variety of human malignant tumors, but in normal tissues only in the testis, ovaries or placenta tissues. The previous study showed that the specific expression of Dazl germ cells was closely related to the differences in the methylation patterns of the gene regulatory region. We found that DAZL is expressed in head and neck tumors, lung cancer and breast cancer in the TCGA database, especially in the latter, but the expression of DAZL in the tumor is still low compared with other classic cancer and reproductive genes, suggesting that the abnormal expression of DAZL in some tumors is likely to be swollen. The overall abnormal hypomethylation of the tumor cell genome is related.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698;R73

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