本文選題：miR-383　切入點(diǎn)：DNA損傷　出處：《中國科學(xué)技術(shù)大學(xué)》2015年博士論文

【摘要】：近年來,不育已儼然成為困擾全球范圍無數(shù)育齡家庭的嚴(yán)峻問題。根據(jù)WHO標(biāo)準(zhǔn),適齡夫妻未采取避孕手段一年時間仍不能受孕則定義為不育。有數(shù)據(jù)顯示,10%-15%的家庭不能通過自然方式受孕,其中男性不育因素占到一半。盡管部分男性不育是由于已知原因造成,如生殖道感染,隱睪,精索靜脈曲張,輸精管堵塞,性功能障礙等等,但是多數(shù)的男性不育都被診斷為未知原因的非梗阻性無精癥(NOA)。而且,NOA病人具有較低的精子獲取率,臨床受孕率以及更高的精子DNA損傷率。因此,開展研究NOA的發(fā)病機(jī)理和潛在的分子機(jī)制能為臨床治療NOA患者提供方向和指導(dǎo)意見。 精子發(fā)生是一個精密調(diào)控的生理過程,發(fā)生在睪丸內(nèi)的曲細(xì)精管,主要包括從未分化的精原干細(xì)胞發(fā)育至高度分化的成熟精子細(xì)胞的過程,包括進(jìn)行有絲分裂的精原細(xì)胞以及進(jìn)行減數(shù)分裂的精母細(xì)胞。在精子發(fā)生后期,減數(shù)分裂之后轉(zhuǎn)錄活性受到抑制。轉(zhuǎn)錄形成的mRNA主要是受到轉(zhuǎn)錄后調(diào)控,其中很重要的因子即是microRNA (miRNA)。miRNA即微小RNA,是一段由19-23個核苷酸序列組成的非編碼RNA,通過自身的種子序列(Seed sequence)與其靶基因的3端非編碼區(qū)域(UTR)進(jìn)行完全或不完全配對結(jié)合,降解其靶基因的mRNA或抑制靶基因翻譯的方式來進(jìn)行基因表達(dá)的調(diào)控。 大量證據(jù)顯示,具有較高的DNA損傷的生殖細(xì)胞導(dǎo)致生精過程的受損,進(jìn)而造成男性不育。我們研究組先前的小分子RNA芯片(micro-array)結(jié)果顯示,microRNA-383(miR-383)在精子成熟抑制(MA)病人睪丸組織中呈顯著下調(diào),并且通過原位雜交和實(shí)時熒光定量PCR技術(shù)發(fā)現(xiàn)了miR-383主要定位表達(dá)于精子發(fā)生早期的精原細(xì)胞和初級精母細(xì)胞。而早期生精細(xì)胞由于有絲分裂和減數(shù)分裂,重組染色體,具有DNA損傷的較高風(fēng)險。然而,miR-383是否參與調(diào)控在精子發(fā)生過程中的DNA損傷,以何種形式調(diào)控?本論文的首要目的就是闡明miR-383在精子發(fā)生過程中DNA損傷的調(diào)控機(jī)理和分子機(jī)制。首先我們通過NOA患者睪丸組織鋪展發(fā)現(xiàn),NOA患者睪丸組織的DNA損傷率是正常對照的3倍左右。另外,通過細(xì)胞免疫熒光和蛋白質(zhì)印記技術(shù)我們檢測了DNA損傷位點(diǎn)的標(biāo)識分子yH2AX,結(jié)果顯示miR-383減少yH2AX聚集點(diǎn)和聚集程度,抑制yH2AX的蛋白水平,而且由于順鉑藥物(cisplatin)能造成細(xì)胞DNA損傷,經(jīng)順鉑藥物處理后,我們發(fā)現(xiàn)miR-383能夠增強(qiáng)人源睪丸胚胎瘤細(xì)胞(NT-2)對順鉑藥物的敏感性,說明miR-383參與DNA損傷途徑。通過利用分子信息學(xué)和熒光素酶報告基因技術(shù),我們發(fā)現(xiàn)了蛋白磷酸酶1核內(nèi)靶向亞基(PNUTS)是miR-383的靶基因,而且經(jīng)介導(dǎo)實(shí)驗(yàn)發(fā)現(xiàn)PNUTS參與到miR-383對yH2AX的調(diào)控途徑。同時,由于miR-383能誘導(dǎo)細(xì)胞周期停滯在細(xì)胞周期的G1期,減少復(fù)制期(S期)細(xì)胞,從而減少yH2AX的表達(dá)。但是通過,基因沉默PNUTS(siPNUTS)發(fā)現(xiàn),siPNUTS并不直接影響細(xì)胞周期。綜上所述,miR-383通過兩條獨(dú)立的通路調(diào)控yH2AX,一方面是通過靶向PNUTS,從而介導(dǎo)yH2AX的生成；另一方面通過抑制了細(xì)胞周期的停滯,從而減少yH2AX的表達(dá)。 另外一方面,導(dǎo)致男性不育的一個重要原因是隱睪,隱睪的發(fā)比率為2-4%而且發(fā)病率逐年上漲。在不發(fā)達(dá)國家中,隱睪的患病率則更為嚴(yán)重。目前臨床上治療隱睪的方法主要是睪丸固定術(shù),醫(yī)生一般要求在6月-2歲之間進(jìn)行手術(shù),以減少不育和睪丸癌癥的風(fēng)險。有研究表明,即使手術(shù)成功,男嬰在成年后患男性不育的概率達(dá)50%以上,隱睪的發(fā)生機(jī)制和對睪丸造成的分子通路仍然沒有明確定論。近年有研究報道隱睪與下丘腦-垂體-性腺軸激素生成,基因INL3/LGR8的突變等因素有關(guān),但是隱睪中microRNA的調(diào)控機(jī)制還鮮有報道。因此本論文也通過建立隱睪小鼠模型,探索研究了]nicroRNA-210(miR-210)在小鼠隱睪模型中的功能。 我們研究發(fā)現(xiàn)在人隱睪患者睪丸組織中,miR-210顯著上升,在小鼠隱睪模型的隱睪側(cè)也有著相同的效應(yīng)。睪丸組織切片免疫熒光實(shí)驗(yàn)顯示,隱睪側(cè)睪丸有明顯的DNA損傷,且曲細(xì)精管生殖細(xì)胞脫落明顯。在體外實(shí)驗(yàn),發(fā)現(xiàn)TNFa能誘導(dǎo)下游NFkB的p65亞基抑制miR-210,對于隱睪中miR-210的上升呈現(xiàn)負(fù)反饋的調(diào)節(jié)。但是miR-210在隱睪中上升的具體生理意義和分子機(jī)制還有待我們進(jìn)一步探索與研究。
[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.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R698.2

【共引文獻(xiàn)】

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