miRNA和遗传因素在男性不育发生中的功能研究

发布时间：2018-06-07 04:38

本文选题：男性不育 + miR-383　；参考：《中国科学技术大学》2014年博士论文

【摘要】：不育困扰着10-15%的育龄夫妇,其中一半的因素来源于男性。尽管有数十种造成男性不育的病因学机制,但大多数男性不育的发生是原发性的。例如,很大比例的男性不育被诊断为不可解释的非梗阻性无精症(NOA)。进而,NOA患者通常具有较低的精子质量和临床受孕率。因此,阐明NOA潜在的分子发病机理将会限制促进男性不育的诊断和治疗。精子发生是从精原干细胞发育到高度分化的精子细胞的过程,这个过程被严格调控。在精子发生减数分裂和单倍体形成时期,转录活性增加,翻译被抑制。这些时期的mRNA翻译抑制主要是通过转录后调控机制调控,其中就包括microRNA(miRNA).我们研究组已经报道microRNA-383(miR-383)在成熟抑制型(MA)男性不育患者的睾丸中显著下调,在本论文的首要研究目的就是阐明miR-383在MA型男性不育发生中潜在的分子机制。我们发现miR-383通过靶向肿瘤抑制因子interferon regulatory factor-1(IRF1)介导抑制细胞增殖、细胞周期G1期停滞和诱导细胞凋亡的效应,导致IRF1的下游靶基因Cyclin D1、CDK2、p21下调,而最终导致pRb磷酸化被抑制。这些结果将导致miR-383通过靶向IRF1、失活pRb而成为细胞增殖的负调因子。异常的miR-383表达可能成为连接男性不育和生殖细胞肿瘤的桥梁。尽管上部分研究发现miR-383在MA型男性不育患者中下调,但miR-383的功能和靶向性是如何被调控的依旧未知。因此在本论文第二部分(第三章)的研究中,我们试图阐述miR-383在精子发生中的调控机理。我们发现FMRP在小鼠睾丸中结合包括miR-383在内的88个miRNAs。FMRP将通过抑制miR-383结合到其靶基因(IRF1(?)CyclinD1)的3’非编码区而提高miR-383诱导的细胞增殖抑制效应。另一方面,在NT-2和GC1(小鼠精原细胞系)中,FMRP的水平也被miR-383直接靶向Cyclin D1而下调。在Fmr1基因敲除小鼠睾丸中,我们发现miR-383表达下调、CDK4表达定位失调、DNA损伤增加等现象,而相似的现象在FMRP下调的MA型男性不育患者的睾丸中也被检测到。因而,FMRP-miR-383在精子发生中可以构成一个潜在的反馈调控回路,FMRP则扮演miR-383功能的负调因子角色。我们的研究成果也表明FMRP-miR-383调控通路的失调可能和MA型男性不育的发生密切相关。另一方面,遗传因素也能影响男性不育。Kallmann综合症(KS)是一个遗传性进行性的失调疾病,症状为低促性腺激素功能减退症(导致不育)和嗅觉缺失／降低。由于在胚胎期的嗅球发育不全,神经内分泌细胞GnRH不能正常的沿着嗅神经纤维从鼻部向前脑迁移。现在已经发现大量的基因均与这种神经元的轴突导向相关,KS也被发现是一种遗传异质性疾病,涉及到多种遗传方式。但是,如今已经发现的致病基因只能解释近三分之一的KS患者的病因,还远远不能完全解释这种疾病,提示了还存在其他基因或调控通路参与到KS的发生中。在本论文的第三部分中,我们报道了一个汉族母系遗传KS家系。这个家系存在两个新发协同突变,分别是KAL1基因146GT突变(p.49CysPhe)线粒体tRNAcysmt.5800AG突变。进一步,线粒体cysteinyl-tRNA途径的失调能显著影响GnRH神经元细胞的体外迁移。而且,线粒体cysteinyl-tRNA转移酶Cars2基因也是调控斑马鱼GnRH3神经元迁移、幼鱼存活和斑马鱼嗅觉功能的关键因素。因此,在这部分我们提出了线粒体在GnRH(?)神经元迁移中起到重要的作用,为KS的遗传病因学提供了新的线索。综上所述,本论文研究结果阐述了miR-383/FMRP反馈调控回路在精原细胞增殖凋亡调控和KAL1/线粒体半胱氨酸tRNA途径在Kallmann综合症中的功能。这些研究成果将为进一步理解精子发生缺陷和GnRH神经元迁移异常的分子调控机制奠定基础。
[Abstract]:Infertile couples of childbearing age in 10-15%, half of which are derived from men. Although there are ten etiological mechanisms that cause male infertility, most male infertility is primary. For example, a large proportion of male infertility is diagnosed as uninterpretable non obstructive azoosinoses (NOA). In turn, the NOA patients usually have more than one. Therefore, clarifying the potential molecular pathogenesis of NOA will limit the diagnosis and treatment of male infertility.
Spermatogenesis is a process from spermatogonial stem cells to highly differentiated spermatozoa. This process is strictly regulated. In the period of meiosis and haploid formation of spermatogenesis, the transcriptional activity is increased, and translation is suppressed. The inhibition of mRNA translation during these periods is mainly regulated by post transcriptional regulation mechanism, including microRNA (miRNA). Our research team has reported that microRNA-383 (miR-383) is significantly down-regulated in the testicles of male infertility patients with mature suppressor type (MA). The primary purpose of this study is to elucidate the potential molecular mechanism of miR-383 in the occurrence of MA male infertility. We found that miR-383 passes through the target tumor suppressor, interferon regulatory factor-1 (IRF), (IRF). 1) mediating inhibition of cell proliferation, stagnation of cell cycle G1 and inducing cell apoptosis, leading to the downstream target gene Cyclin D1, CDK2, p21 downregulation, and ultimately leading to the inhibition of pRb phosphorylation. These results will lead to miR-383 through target IRF1, deactivation pRb and become a negative regulator of cell proliferation. Abnormal miR-383 expression may become a link. A bridge between male infertility and germ cell tumors.
Although the previous study found that miR-383 was downregulated in MA type male infertility, the function and targeting of miR-383 remained unknown. Therefore, in the second part of this paper (third chapter), we tried to explain the mechanism of miR-383 in the regulation of spermatogenesis. We found that FMRP in mouse testis consists of miR-3 88 miRNAs.FMRP, 83, will increase the inhibitory effect of miR-383 induced cell proliferation by inhibiting the 3 'non coding region of the miR-383 binding to its target gene (IRF1 (?) CyclinD1). On the other hand, in NT-2 and GC1 (mouse spermatogonial cell lines), the level of FMRP is also downregulated by miR-383 directly to Cyclin D1. We found that the downregulation of miR-383, the imbalance of CDK4 expression, the increase of DNA damage, and similar phenomena were detected in the testicles of the MA type male sterility patients with the downregulation of FMRP. Therefore, FMRP-miR-383 could form a potential feedback loop in spermatogenesis, and FMRP acts as a negative factor role in miR-383 function. Our findings also suggest that the imbalance of FMRP-miR-383 regulation pathway may be closely related to the occurrence of MA type male infertility.
On the other hand, genetic factors can also affect male infertility.Kallmann syndrome (KS) as a hereditary progressive disorder with low gonadotropin hypogonadism (leading to infertility) and loss of smell / decrease. The neuroendocrine cell GnRH is not normal along the olfactory nerve fiber due to the dysplasia of the olfactory bulb during the embryonic period. A large number of genes have been found to be associated with the axon direction of this neuron, and KS is also found to be a genetic heterozygous disease that involves a variety of genetic patterns. However, the pathogenetic genes that have now been discovered can only explain the causes of nearly 1/3 of KS patients and are far from fully explaining the disease, There are other genes or regulatory pathways involved in the occurrence of KS. In the third part of this paper, we report a family of Han maternal hereditary KS family. There are two new synergistic mutations in this family, the mitochondrial tRNAcysmt.5800AG mutation of the KAL1 gene 146GT mutation (p.49CysPhe). Further, mitochondrial cysteinyl-tRNA The maladjustment of the pathway can significantly affect the migration of GnRH neuron cells in vitro. Moreover, the mitochondrial cysteinyl-tRNA transferase Cars2 gene is also a key factor in regulating the migration of GnRH3 neurons in zebrafish, the survival of young fish and the olfactory function of zebrafish. Therefore, we have proposed that the cord body plays an important role in the migration of GnRH (?) neurons in this part. It is used to provide new clues for the genetic etiology of KS.
To sum up, the results of this study illustrate the regulation of miR-383/FMRP feedback control loop on spermatogonial cell proliferation and apoptosis and the function of KAL1/ mitochondrial cysteine tRNA pathway in Kallmann syndrome. These results will lay the foundation for further understanding the molecular regulation mechanism of spermatogenesis defects and the abnormal migration of GnRH neurons.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R698.2

【共引文献】