本文選題：男性不育 + miR-383　； 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2014年博士論文

【摘要】：不育困擾著10-15%的育齡夫婦,其中一半的因素來(lái)源于男性。盡管有數(shù)十種造成男性不育的病因?qū)W機(jī)制,但大多數(shù)男性不育的發(fā)生是原發(fā)性的。例如,很大比例的男性不育被診斷為不可解釋的非梗阻性無(wú)精癥(NOA)。進(jìn)而,NOA患者通常具有較低的精子質(zhì)量和臨床受孕率。因此,闡明NOA潛在的分子發(fā)病機(jī)理將會(huì)限制促進(jìn)男性不育的診斷和治療。 精子發(fā)生是從精原干細(xì)胞發(fā)育到高度分化的精子細(xì)胞的過(guò)程,這個(gè)過(guò)程被嚴(yán)格調(diào)控。在精子發(fā)生減數(shù)分裂和單倍體形成時(shí)期,轉(zhuǎn)錄活性增加,翻譯被抑制。這些時(shí)期的mRNA翻譯抑制主要是通過(guò)轉(zhuǎn)錄后調(diào)控機(jī)制調(diào)控,其中就包括microRNA(miRNA).我們研究組已經(jīng)報(bào)道m(xù)icroRNA-383(miR-383)在成熟抑制型(MA)男性不育患者的睪丸中顯著下調(diào),在本論文的首要研究目的就是闡明miR-383在MA型男性不育發(fā)生中潛在的分子機(jī)制。我們發(fā)現(xiàn)miR-383通過(guò)靶向腫瘤抑制因子interferon regulatory factor-1(IRF1)介導(dǎo)抑制細(xì)胞增殖、細(xì)胞周期G1期停滯和誘導(dǎo)細(xì)胞凋亡的效應(yīng),導(dǎo)致IRF1的下游靶基因Cyclin D1、CDK2、p21下調(diào),而最終導(dǎo)致pRb磷酸化被抑制。這些結(jié)果將導(dǎo)致miR-383通過(guò)靶向IRF1、失活pRb而成為細(xì)胞增殖的負(fù)調(diào)因子。異常的miR-383表達(dá)可能成為連接男性不育和生殖細(xì)胞腫瘤的橋梁。 盡管上部分研究發(fā)現(xiàn)miR-383在MA型男性不育患者中下調(diào),但miR-383的功能和靶向性是如何被調(diào)控的依舊未知。因此在本論文第二部分(第三章)的研究中,我們?cè)噲D闡述miR-383在精子發(fā)生中的調(diào)控機(jī)理。我們發(fā)現(xiàn)FMRP在小鼠睪丸中結(jié)合包括miR-383在內(nèi)的88個(gè)miRNAs。FMRP將通過(guò)抑制miR-383結(jié)合到其靶基因(IRF1(?)CyclinD1)的3’非編碼區(qū)而提高miR-383誘導(dǎo)的細(xì)胞增殖抑制效應(yīng)。另一方面,在NT-2和GC1(小鼠精原細(xì)胞系)中,FMRP的水平也被miR-383直接靶向Cyclin D1而下調(diào)。在Fmr1基因敲除小鼠睪丸中,我們發(fā)現(xiàn)miR-383表達(dá)下調(diào)、CDK4表達(dá)定位失調(diào)、DNA損傷增加等現(xiàn)象,而相似的現(xiàn)象在FMRP下調(diào)的MA型男性不育患者的睪丸中也被檢測(cè)到。因而,FMRP-miR-383在精子發(fā)生中可以構(gòu)成一個(gè)潛在的反饋調(diào)控回路,FMRP則扮演miR-383功能的負(fù)調(diào)因子角色。我們的研究成果也表明FMRP-miR-383調(diào)控通路的失調(diào)可能和MA型男性不育的發(fā)生密切相關(guān)。 另一方面,遺傳因素也能影響男性不育。Kallmann綜合癥(KS)是一個(gè)遺傳性進(jìn)行性的失調(diào)疾病,癥狀為低促性腺激素功能減退癥(導(dǎo)致不育)和嗅覺(jué)缺失／降低。由于在胚胎期的嗅球發(fā)育不全,神經(jīng)內(nèi)分泌細(xì)胞GnRH不能正常的沿著嗅神經(jīng)纖維從鼻部向前腦遷移�，F(xiàn)在已經(jīng)發(fā)現(xiàn)大量的基因均與這種神經(jīng)元的軸突導(dǎo)向相關(guān),KS也被發(fā)現(xiàn)是一種遺傳異質(zhì)性疾病,涉及到多種遺傳方式。但是,如今已經(jīng)發(fā)現(xiàn)的致病基因只能解釋近三分之一的KS患者的病因,還遠(yuǎn)遠(yuǎn)不能完全解釋這種疾病,提示了還存在其他基因或調(diào)控通路參與到KS的發(fā)生中。在本論文的第三部分中,我們報(bào)道了一個(gè)漢族母系遺傳KS家系。這個(gè)家系存在兩個(gè)新發(fā)協(xié)同突變,分別是KAL1基因146GT突變(p.49CysPhe)線粒體tRNAcysmt.5800AG突變。進(jìn)一步,線粒體cysteinyl-tRNA途徑的失調(diào)能顯著影響GnRH神經(jīng)元細(xì)胞的體外遷移。而且,線粒體cysteinyl-tRNA轉(zhuǎn)移酶Cars2基因也是調(diào)控斑馬魚(yú)GnRH3神經(jīng)元遷移、幼魚(yú)存活和斑馬魚(yú)嗅覺(jué)功能的關(guān)鍵因素。因此,在這部分我們提出了線粒體在GnRH(?)神經(jīng)元遷移中起到重要的作用,為KS的遺傳病因?qū)W提供了新的線索。 綜上所述,本論文研究結(jié)果闡述了miR-383/FMRP反饋調(diào)控回路在精原細(xì)胞增殖凋亡調(diào)控和KAL1/線粒體半胱氨酸t(yī)RNA途徑在Kallmann綜合癥中的功能。這些研究成果將為進(jìn)一步理解精子發(fā)生缺陷和GnRH神經(jīng)元遷移異常的分子調(diào)控機(jī)制奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R698.2

【共引文獻(xiàn)】

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本文編號(hào)：1989820