MicroRNA在男性不育中的功能研究

发布时间：2018-04-01 14:05

本文选题：miR-383　切入点：DNA损伤　出处：《中国科学技术大学》2015年博士论文

【摘要】：近年来,不育已俨然成为困扰全球范围无数育龄家庭的严峻问题。根据WHO标准,适龄夫妻未采取避孕手段一年时间仍不能受孕则定义为不育。有数据显示,10%-15%的家庭不能通过自然方式受孕,其中男性不育因素占到一半。尽管部分男性不育是由于已知原因造成,如生殖道感染,隐睾,精索静脉曲张,输精管堵塞,性功能障碍等等,但是多数的男性不育都被诊断为未知原因的非梗阻性无精症(NOA)。而且,NOA病人具有较低的精子获取率,临床受孕率以及更高的精子DNA损伤率。因此,开展研究NOA的发病机理和潜在的分子机制能为临床治疗NOA患者提供方向和指导意见。精子发生是一个精密调控的生理过程,发生在睾丸内的曲细精管,主要包括从未分化的精原干细胞发育至高度分化的成熟精子细胞的过程,包括进行有丝分裂的精原细胞以及进行减数分裂的精母细胞。在精子发生后期,减数分裂之后转录活性受到抑制。转录形成的mRNA主要是受到转录后调控,其中很重要的因子即是microRNA (miRNA)。miRNA即微小RNA,是一段由19-23个核苷酸序列组成的非编码RNA,通过自身的种子序列(Seed sequence)与其靶基因的3端非编码区域(UTR)进行完全或不完全配对结合,降解其靶基因的mRNA或抑制靶基因翻译的方式来进行基因表达的调控。大量证据显示,具有较高的DNA损伤的生殖细胞导致生精过程的受损,进而造成男性不育。我们研究组先前的小分子RNA芯片(micro-array)结果显示,microRNA-383(miR-383)在精子成熟抑制(MA)病人睾丸组织中呈显著下调,并且通过原位杂交和实时荧光定量PCR技术发现了miR-383主要定位表达于精子发生早期的精原细胞和初级精母细胞。而早期生精细胞由于有丝分裂和减数分裂,重组染色体,具有DNA损伤的较高风险。然而,miR-383是否参与调控在精子发生过程中的DNA损伤,以何种形式调控?本论文的首要目的就是阐明miR-383在精子发生过程中DNA损伤的调控机理和分子机制。首先我们通过NOA患者睾丸组织铺展发现,NOA患者睾丸组织的DNA损伤率是正常对照的3倍左右。另外,通过细胞免疫荧光和蛋白质印记技术我们检测了DNA损伤位点的标识分子yH2AX,结果显示miR-383减少yH2AX聚集点和聚集程度,抑制yH2AX的蛋白水平,而且由于顺铂药物(cisplatin)能造成细胞DNA损伤,经顺铂药物处理后,我们发现miR-383能够增强人源睾丸胚胎瘤细胞(NT-2)对顺铂药物的敏感性,说明miR-383参与DNA损伤途径。通过利用分子信息学和荧光素酶报告基因技术,我们发现了蛋白磷酸酶1核内靶向亚基(PNUTS)是miR-383的靶基因,而且经介导实验发现PNUTS参与到miR-383对yH2AX的调控途径。同时,由于miR-383能诱导细胞周期停滞在细胞周期的G1期,减少复制期(S期)细胞,从而减少yH2AX的表达。但是通过,基因沉默PNUTS(siPNUTS)发现,siPNUTS并不直接影响细胞周期。综上所述,miR-383通过两条独立的通路调控yH2AX,一方面是通过靶向PNUTS,从而介导yH2AX的生成；另一方面通过抑制了细胞周期的停滞,从而减少yH2AX的表达。另外一方面,导致男性不育的一个重要原因是隐睾,隐睾的发比率为2-4%而且发病率逐年上涨。在不发达国家中,隐睾的患病率则更为严重。目前临床上治疗隐睾的方法主要是睾丸固定术,医生一般要求在6月-2岁之间进行手术,以减少不育和睾丸癌症的风险。有研究表明,即使手术成功,男婴在成年后患男性不育的概率达50%以上,隐睾的发生机制和对睾丸造成的分子通路仍然没有明确定论。近年有研究报道隐睾与下丘脑-垂体-性腺轴激素生成,基因INL3/LGR8的突变等因素有关,但是隐睾中microRNA的调控机制还鲜有报道。因此本论文也通过建立隐睾小鼠模型,探索研究了]nicroRNA-210(miR-210)在小鼠隐睾模型中的功能。我们研究发现在人隐睾患者睾丸组织中,miR-210显著上升,在小鼠隐睾模型的隐睾侧也有着相同的效应。睾丸组织切片免疫荧光实验显示,隐睾侧睾丸有明显的DNA损伤,且曲细精管生殖细胞脱落明显。在体外实验,发现TNFa能诱导下游NFkB的p65亚基抑制miR-210,对于隐睾中miR-210的上升呈现负反馈的调节。但是miR-210在隐睾中上升的具体生理意义和分子机制还有待我们进一步探索与研究。
[Abstract]:In recent years, infertility has become serious problems worldwide. Numerous childbearing age family according to the WHO standard, school-age couples did not take contraceptive means a year is still not pregnant is defined as infertility. The data show that the 10%-15% family can not be the natural way of conception, including male infertility factors accounted for half. Although some male infertility is due to the known reasons, such as reproductive tract infection, cryptorchidism, varicocele, blockage of the vas deferens, sexual dysfunction and so on, but most of the male infertility were diagnosed as non obstructive azoospermia of unknown origin (NOA). Moreover, NOA patients have lower sperm extraction rate, clinical pregnancy rate and higher sperm DNA damage rate. Therefore, to carry out the molecular mechanism of pathogenesis of NOA and the potential to provide direction and guidance for the clinical treatment of patients with NOA.
Spermatogenesis is a physiological process of precision control, occurs in the testis seminiferous tube consists primarily of undifferentiated spermatogonial stem cells developed into highly differentiated mature sperm cells, including mitosis in spermatogonia and spermatocyte meiosis. During late stages of spermatogenesis. After meiosis transcriptional activity was inhibited. The transcription mRNA formed mainly by post transcriptional regulation, which is an important factor that is microRNA (miRNA).MiRNA RNA is a small, composed of 19-23 nucleotides encoding the non RNA, through its seed sequence (Seed sequence) 3 non encoding region and its end the target gene (UTR) of complete or incomplete pairing, regulation of its target genes mRNA degradation or inhibit translation of target genes of gene expression.
A lot of evidence that DNA damage of germ cells with high impaired spermatogenesis, causing male infertility. We study the small molecule RNA chipset previously (micro-array) results showed that microRNA-383 (miR-383) in sperm maturation inhibition (MA) of testicular tissue in patients was significantly reduced, and by in situ hybridization and real-time fluorescence quantitative PCR miR-383 was found mainly in the early stage of spermatogenesis spermatogonia and primary spermatocytes and early spermatogenic cells by mitosis and meiosis, chromosome recombination, DNA has higher risk of injury. However, whether miR-383 is involved in regulation of DNA damage in the process of spermatogenesis, regulation what form? The primary purpose of this paper is to clarify the miR-383 in sperm DNA damage mechanism and molecular mechanism in the process. First we through the NOA patients with testicular tissue Spreading, testicular tissue of patients with NOA DNA damage rate is about 3 times higher than that in normal control. In addition, by immunofluorescence and Western blot technique we detected the yH2AX marker of DNA damage sites. The results showed that miR-383 reduced yH2AX accumulation point and the degree of aggregation, inhibition of yH2AX protein level, but also because of cisplatin (cisplatin) can cause DNA damage of cells, after cisplatin treatment, we found that miR-383 can enhance human testicular embryonal carcinoma cells (NT-2) sensitivity to cisplatin treatment, suggesting that miR-383 is involved in DNA damage pathway. By the use of molecular information and luciferase assay, we found that the protein phosphatase 1 nuclear targeting subunit (PNUTS) is the target gene of miR-383, and the results showed that PNUTS mediated regulation of yH2AX pathway involved in miR-383. At the same time, because miR-383 can induce cell cycle arrest in cell cycle During the G1 period, reduce the replication stage (stage S) cells, thereby reducing the expression of yH2AX. But by PNUTS gene silencing (siPNUTS) found that siPNUTS does not directly affect the cell cycle. To sum up, miR-383 through two separate pathways in yH2AX, on the one hand is by targeting PNUTS, which mediates yH2AX generation on the other hand; through inhibiting the cell cycle arrest, thereby reducing the expression of yH2AX.
On the other hand, an important cause of male infertility is cryptorchidism, cryptorchidism hair ratio is 2-4% and the incidence rate is rising year by year. In the less developed countries, the prevalence of cryptorchidism is more serious. At present the clinical treatment of cryptorchidism is orchiorrhaphy, doctors generally require surgery in June at the age of -2 between, to reduce the risk of infertility and testicular cancer. Studies have shown that even if the operation is successful, the baby is more than 50% in the probability of adult male infertility, cryptorchidism on testicular pathogenesis and the cause of molecular pathway is still no clear conclusion. Recent studies have reported cryptorchidism and hypothalamic pituitary gonadal hormone production, mutation the factors such as INL3/LGR8 gene, but the regulation mechanism of microRNA in cryptorchidism is rarely reported. Therefore, this paper through the establishment of cryptorchidism mouse model, explore and study the]nicroRNA-210 (miR-210) in small The function of the rat cryptorchidism model.
Our study found that people in testicular tissue of patients with cryptorchidism, miR-210 increased significantly, also have the same effect in the cryptorchid mouse cryptorchidism model. Showed testis tissue sections by immunofluorescence experiments, cryptorchid testicular injury was DNA, and the seminiferous tubule germ cell shedding significantly. In vitro experiment, found p65 TNFa can induce downstream inhibition of NFkB miR-210, miR-210 for the rose cryptorchidism negative feedback regulation. But miR-210 increased in cryptorchidism in the specific physiological significance and molecular mechanism still needs further exploration and research of us.

【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】：2015
【分类号】：R698.2

【共引文献】