【摘要】：第一部分MicroRNA-22-3p在原發(fā)性卵巢功能不全患者血漿中顯著下調(diào)及意義背景：長(zhǎng)度為21-24核苷酸的非編碼RNA被稱為microRNA (miRNA),廣泛存在于人體各種組織和體液中,在轉(zhuǎn)錄后水平調(diào)控基因表達(dá)。大量研究證實(shí)miRNAs不僅可以作為診斷多種癌癥、糖尿病等的有效標(biāo)志物,在疾病發(fā)病過(guò)程中發(fā)揮重要作用；還可以影響生殖細(xì)胞增殖和凋亡,調(diào)控排卵及黃素化部分關(guān)鍵過(guò)程,參與卵巢功能的多個(gè)方面。目前關(guān)于niRNAs在POI疾病的研究較少,尚不能全面闡明niRNAs在POI患者中的變化及作用。目的：本研究旨在通過(guò)μParafloTM MicroRNA芯片技術(shù)篩選POI患者血漿中差異表達(dá)的miRNAs,并在較大樣本人群中驗(yàn)證明確miRNAs在POI患者血漿中的表達(dá)譜及其臨床作用。方法：募集2012年9月至2013年12月就診于山東大學(xué)附屬生殖醫(yī)院的中國(guó)漢族POI患者140例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對(duì)照女性140例,留取新鮮血漿。首先通過(guò)μParafloTM MicroRNA芯片技術(shù)在10例POI患者和10例對(duì)照女性血漿中篩選差異表達(dá)的miRNAs,選擇差異顯著的miRNAs在260例獨(dú)立樣本中進(jìn)行驗(yàn)證。結(jié)果：芯片篩選出差異表達(dá)miRNAs共51個(gè),其中22個(gè)在POI患者血漿中上調(diào),29個(gè)下調(diào)。選擇表達(dá)豐度較高的9個(gè)差異表達(dá)miRNAs (let-7b-5p、let-7c、 miR-15b-5p、miR-22-3p、miR-23a-3p、miR-23b-3p、miR-24-3p、miR-151a-5p和miR-151b)在100例獨(dú)立樣本中驗(yàn)證,發(fā)現(xiàn)niR-22-3p在POI患者血漿中表達(dá)量下降,其余8個(gè)miRNAs在兩組人群表達(dá)無(wú)顯著差異。隨后我們?cè)?60例樣本中驗(yàn)證miR-22-3p,結(jié)果顯示miR-22-3p在POI患者血漿中表達(dá)顯著降低(差異倍數(shù)：0.74, P0.05); ROC (receiver operating characteristic)分析提示]miR-22-3p區(qū)分POI患者與對(duì)照人群的曲線下面積(Area Under Curve, AUC)為0.668；95%confidence interval (CI):0.602-0.733;截?cái)嘀?cut off value)為0.607,此截?cái)嘀祬^(qū)分POI患者的敏感性和特異性分別是75.4%和54.6%�；貧w分析表明miR-22-3p是POI保護(hù)性因素(OR值：0.766,95%CI：0.643-0.912),與血清FSH水平負(fù)相關(guān)(R2=0.687,P0.05)。結(jié)論：本研究在POI患者血漿中篩選差異表達(dá)的miRNAs,發(fā)現(xiàn)miR-22-3p在POI患者表達(dá)量顯著降低,與血清FSH水平負(fù)相關(guān)。提示miR-22-3p可能反映卵巢儲(chǔ)備功能,是POI病理過(guò)程的一個(gè)結(jié)果。第二部分MicroRNA-379-5p調(diào)控PARP1、XRCC6在生化異常期原發(fā)性卵巢功能不全中的作用和機(jī)制研究背景：原發(fā)性卵巢功能不全病因高度異質(zhì),卵子發(fā)生障礙和卵泡耗竭加速是目前POI兩種主要的致病機(jī)制假說(shuō)。顆粒細(xì)胞是包繞卵母細(xì)胞的體細(xì)胞,與卵母細(xì)胞之間存在復(fù)雜的調(diào)控網(wǎng)絡(luò),大量研究指出顆粒細(xì)胞增殖、分化、侵襲等功能異常是卵巢卵泡耗竭的原因之一,而miRNAs可能在其中發(fā)揮重要作用。已有動(dòng)物實(shí)驗(yàn)和體外實(shí)驗(yàn)表明miRNAs可以影響顆粒細(xì)胞增殖、凋亡及激素合成功能,但尚無(wú)POI患者顆粒細(xì)胞中miRNAs表達(dá)譜研究及功能研究。目的：本課題通過(guò)niRCURY?芯片技術(shù)篩選生化異常期POI患者顆粒細(xì)胞中差異表達(dá)的miRNAs;結(jié)合表達(dá)譜芯片結(jié)果,針對(duì)差異表達(dá)的miRNAs及其可能的靶基因進(jìn)行功能實(shí)驗(yàn),研究其對(duì)顆粒細(xì)胞生物學(xué)功能的影響和作用機(jī)制,從表觀遺傳角度探討非編碼RNA參與POI發(fā)生發(fā)展過(guò)程的分子機(jī)理。方法：募集2013年9月至2014年12月就診于山東大學(xué)附屬生殖醫(yī)院的中國(guó)漢族生化異常階段POI患者50例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對(duì)照女性50例,留取顆粒細(xì)胞。通過(guò)miRCURY?芯片技術(shù)篩選差異表達(dá)的miRNAs;與表達(dá)譜芯片聯(lián)合分析選擇差異最顯著的miR-379-5p和其可能的靶基因PARP1、 XRCC6,通過(guò)Real-Time PCR在獨(dú)立樣本顆粒細(xì)胞中驗(yàn)證它們的表達(dá)趨勢(shì)；在顆粒細(xì)胞系中過(guò)表達(dá)miR-379-5p,分別在未處理狀態(tài)和紫外線(UV)和雙氧水(H202)損傷后通過(guò)MTT、CCK8和EdU實(shí)驗(yàn)驗(yàn)證miR-379-5p對(duì)顆粒細(xì)胞增殖能力及損傷修復(fù)功能的影響；雙熒光素酶報(bào)告系統(tǒng)實(shí)驗(yàn)等驗(yàn)證niR-379-5p對(duì)靶基因PARP1和XRCC6的調(diào)控及機(jī)制；沉默顆粒細(xì)胞中內(nèi)源性PARP1和XRCC6,重復(fù)上述實(shí)驗(yàn),明確miR-379-5p降調(diào)PARP1和XRCC6,對(duì)顆粒細(xì)胞增殖和DNA損傷修復(fù)功能的影響和作用機(jī)制。結(jié)果：芯片篩選出差異表達(dá)miRNAs共75條。我們對(duì)miRNA芯片和表達(dá)譜芯片結(jié)果進(jìn)行聯(lián)合分析,鎖定miR-379-5p和它可能的靶基因PARPU XRCC60在60例獨(dú)立樣本中證實(shí)miR-379-5p在生化異常期POI患者顆粒細(xì)胞中顯著上調(diào),PARPU XRCC6在POI患者顆粒細(xì)胞中顯著下調(diào)。過(guò)表達(dá)顆粒細(xì)胞中miR-379-5p可以顯著抑制KGN細(xì)胞增殖,其增殖抑制作用呈時(shí)間依賴性；在UV/H2O2損傷后,過(guò)表達(dá)niR-379-5p組KGN細(xì)胞增殖抑制作用尤其顯著。Luciferase和western blot實(shí)驗(yàn)證實(shí)miR-379-5p通過(guò)與PARPUXRCC6的3'UTR區(qū)域結(jié)合,降調(diào)PARPU XRCC6基因。特異性沉默內(nèi)源性PARPU XRCC6基因?qū)GN細(xì)胞生物學(xué)行為可以產(chǎn)生與過(guò)表達(dá)miR-379-5p類似的作用,包括抑制KGN細(xì)胞增殖、UV/H2O2損傷后較低的細(xì)胞存活率和顯著的增殖抑制作用等。結(jié)論：本研究首次發(fā)現(xiàn)POI患者顆粒細(xì)胞中顯著上調(diào)的miR-379-5p可能通過(guò)降調(diào)PARPU XRCC6影響顆粒細(xì)胞增殖及損傷修復(fù)功能,參與POI疾病進(jìn)程。第三部分長(zhǎng)鏈非編碼RNA在生化異常期原發(fā)性卵巢功能不全中的初步研究背景：長(zhǎng)鏈非編碼RNA (long noncoding RNA, IncRNA)是由RNA聚合酶Ⅱ轉(zhuǎn)錄形成、長(zhǎng)度超過(guò)200nt的RNA分子,具有保守的剪接點(diǎn)和內(nèi)含子,在組織和細(xì)胞中有特異的表達(dá)模式。LncRNAs可以參與染色體重塑、基因轉(zhuǎn)錄的激活和干擾、以及核內(nèi)運(yùn)輸?shù)戎匾{(diào)控過(guò)程,通過(guò)表觀遺傳沉默、轉(zhuǎn)錄和轉(zhuǎn)錄后水平調(diào)節(jié)等方式對(duì)基因表達(dá)進(jìn)行調(diào)控,近年來(lái),逐漸成為生物醫(yī)學(xué)的研究熱點(diǎn)。已有研究證實(shí)lncRNAs在癌癥和神經(jīng)系統(tǒng)疾病等人類重大疾病過(guò)程中發(fā)揮了重要的調(diào)控作用,但目前已經(jīng)明確功能的lncRNAs僅有數(shù)十條,而在生殖系統(tǒng)疾病領(lǐng)域,尚無(wú)相關(guān)研究報(bào)道。目的：本研究通過(guò)芯片技術(shù)篩選并驗(yàn)證生化異常階段POI患者顆粒細(xì)胞中差異表達(dá)的lncRNAs,初步探討lncRNAs在POI患者顆粒細(xì)胞中的表達(dá)情況。方法：募集2013年9月至2014年12月就診的中國(guó)漢族生化異常階段POI患者50例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對(duì)照女性50例,留取顆粒細(xì)胞。通過(guò)LncRNA芯片技術(shù)在10例POI患者與10例對(duì)照女性顆粒細(xì)胞中篩選差異表達(dá)的lncRNAs和mRNAs;根據(jù)表達(dá)譜芯片Gene ontology和KEGG pathway分析結(jié)果,對(duì)差異表達(dá)的lncRNAs和mRNAs進(jìn)行共表達(dá)網(wǎng)絡(luò)分析(co-expression networks),按照相對(duì)表達(dá)豐度、變化倍數(shù)及序列信息,選擇處于核心位置、與重要且差異表達(dá)的基因存在相關(guān)關(guān)系的14條lncRNAs在60例獨(dú)立樣本中進(jìn)行驗(yàn)證。結(jié)果：芯片結(jié)果發(fā)現(xiàn)差異表達(dá)lncRNAs序列677條。Real-Time PCR實(shí)驗(yàn)證實(shí)其中：TCONS 00010009、TCONS 00014547、NR 040662.1、ENST00000520306、ENST00000420313、ENST00000526225在POI患者顆粒細(xì)胞中顯著下調(diào)。生物信息學(xué)分析表明它們?nèi)繉儆趌incRNAs,其周圍均分布有許多功能重要的編碼基因,尤其DNA損傷修復(fù)基因XRCC4和MSH5分別位于TCONS_00010009和NR 040662附近不足300kb處,提示我們這些差異表達(dá)的lincRNAs可能通過(guò)調(diào)控周圍的共表達(dá)基因參與POI疾病。結(jié)論：本研究首次通過(guò)Arraystar Human IncRNA芯片對(duì)顆粒細(xì)胞OncRNAs特征進(jìn)行初步研究。生化異常階段POI患者顆粒細(xì)胞中存在較多差異表達(dá)的LncRNA,可能與疾病相關(guān)。
[Abstract]:The first part is the significant down-regulation and significance of MicroRNA-22-3p in the plasma of patients with primary ovarian insufficiency: the non coded RNA with a length of 21-24 nucleotides is called microRNA (miRNA), which widely exists in various tissues and body fluids of the human body and regulates gene expression at post transcriptional level. A large number of studies have confirmed that miRNAs can not only be used as a diagnostic multiple. The effective markers of cancer and diabetes, such as diabetes, play an important role in the pathogenesis of disease; it can also affect the proliferation and apoptosis of germ cells, regulate the key processes of ovulation and yellowing, and participate in many aspects of ovarian function. At present, there are few studies on niRNAs in the disease of POI, and there is no comprehensive explanation of niRNAs in POI patients. Objective: the purpose of this study was to screen the differential expression of miRNAs in the plasma of POI patients by micron ParafloTM MicroRNA chip technology, and to verify the expression and clinical effect of miRNAs in the plasma of POI patients in a large sample population. Methods: to collect the Reproductive Medicine Affiliated to the Shandong University from September 2012 to December 2013. 140 cases of POI patients in Chinese Han nationality, 140 cases of normal control women with endocrine and fallopian tube factors, 140 cases of fresh plasma were left. The differential expression of miRNAs was screened in 10 cases of POI patients and 10 cases of control female plasma by micron MicroRNA chip technique. The difference of significant miRNAs was selected in 260 independent samples. Results: the chip screened a total of 51 differentially expressed miRNAs, of which 22 were up-regulated and 29 down-regulated in the plasma of POI patients. 9 differential expressions of miRNAs (let-7b-5p, Let-7c, miR-15b-5p, miR-22-3p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-151a-5p and miR-151b) were verified in 100 independent samples. The expression of R-22-3p in the plasma of POI patients decreased and the other 8 miRNAs expressed no significant difference in the two groups. Then we tested the miR-22-3p in 160 samples. The results showed that the expression of miR-22-3p in the plasma of POI patients was significantly reduced (the difference Multiplier: 0.74, P0.05); ROC (receiver operating characteristic) analysis suggested]miR-22-3p area The area under the curve (Area Under Curve, AUC) in the POI patients and the control group was 0.668, 95%confidence interval (CI): 0.602-0.733, and the truncated value (cut off value) was 0.607. 643-0.912) and negative correlation with serum FSH level (R2=0.687, P0.05). Conclusion: This study screened the differential expression of miRNAs in the plasma of POI patients. It was found that the expression of miR-22-3p in POI patients was significantly lower and negatively correlated with the level of serum FSH. It suggested that miR-22-3p may reflect the ovarian reserve power, which is a result of POI pathological process. Second part MicroRNA. -379-5p regulates the role and mechanism of PARP1 and XRCC6 in primary ovarian dysfunction during biochemical abnormal stages: the high heterogeneity of the etiology of primary ovarian dysfunction, the disorder of oogenesis and the acceleration of follicle exhaustion are the two main pathogenesis hypothesis of POI. Granulosa cells are somatic cells wrapped around oocytes and oocytes. There is a complex regulatory network. A large number of studies have pointed out that the proliferation, differentiation and invasion of granulosa cells are one of the reasons for ovarian follicle depletion, and miRNAs may play an important role in it. Animal experiments and in vitro experiments have shown that miRNAs can affect granulosa cell proliferation, apoptosis and hormone synthesis, but there is no POI patient yet. Study and function study of miRNAs expression in granulosa cells. Objective: to screen the differential expression of miRNAs in granulosa cells of patients with POI in biochemical abnormal phase by niRCURY chip technology, and to study the granulosa cell biology by combining the results of the expression spectrum chip to the differentially expressed miRNAs and its possible target genes. The effect and mechanism of function, from the epigenetic point of view, to explore the molecular mechanism of the non coded RNA participating in the development of POI. Methods: 50 cases of POI patients were collected from September 2013 to December 2014 in the affiliated reproductive Hospital of Shandong University. 50 women compared with 50 women, retained granular cells. The differentially expressed miRNAs was screened by miRCURY chip technology; the most significant difference was selected with the expression spectrum chip to select the most significant miR-379-5p and its possible target gene PARP1, XRCC6. The expression trend was verified by Real-Time PCR in the independent sample granulosa cells, and the table in the granular cell line was overexpressed. The effects of miR-379-5p on the proliferation and repair function of granulosa cells were verified by MTT, CCK8 and EdU experiments after the untreated state and ultraviolet (UV) and hydrogen peroxide (H202) damage, respectively. The regulation and mechanism of niR-379-5p on the target gene PARP1 and XRCC6 were verified by the double luciferase reporter system experiment, and the silencing of granular cells was verified by the double luciferase reporter system experiment. Endogenous PARP1 and XRCC6, repeat the above experiments to determine the effect and mechanism of miR-379-5p downfall PARP1 and XRCC6 on the proliferation of granulosa cells and the function of DNA damage repair. Results: the chip screened a total of 75 differentially expressed miRNAs strips. We combined the results of miRNA chip and expression spectrum chip, locked miR-379-5p and its possible target. In 60 independent samples, gene PARPU XRCC60 showed that miR-379-5p was significantly up-regulated in granulosa cells of POI patients with abnormal biochemical phase, and PARPU XRCC6 was significantly down-regulated in granulosa cells of POI patients. MiR-379-5p could significantly inhibit the proliferation of KGN cells in the overexpressed granulosa cells, and its proliferation inhibition was time dependent; after UV/H2O2 damage, a table was overexpressed. The proliferation inhibition effect of KGN cells in the niR-379-5p group was particularly significant.Luciferase and Western blot experiments confirmed that miR-379-5p could reduce the PARPU XRCC6 gene by binding to the 3'UTR region of PARPUXRCC6. N cell proliferation, lower cell survival rate and significant proliferation inhibition after UV/H2O2 injury. Conclusion: This study first found that the significant up regulation of miR-379-5p in granulosa cells of POI patients may be involved in the function of granulosa cell proliferation and injury repair by lowering the modulation of PARPU XRCC6 in the POI disease process. The third part of the long chain non coded RNA is in the process. Primary research background in primary ovarian dysfunction in biochemical abnormal phase: long chain non coded RNA (long noncoding RNA, IncRNA) is transcribed by RNA polymerase II, with a RNA molecule in length exceeding 200nt, with a conservative splicing point and intron, and a specific expression pattern in tissues and cells that.LncRNAs can be involved in chromosome remodeling. The activation and interference of gene transcription, as well as important regulatory processes such as intra nuclear transport, regulate gene expression through epigenetic silence, transcriptional and post transcriptional regulation. In recent years, it has gradually become a research hotspot in biomedicine. LncRNAs has been proved to be in the process of human major diseases such as cancer and nervous system diseases. It has played an important role in regulation and control, but there are only dozens of lncRNAs clearly defined functions, and there are no related reports in the field of reproductive system disease. Objective: This study screened and verified the differential expression of lncRNAs in granulosa cells of patients with POI in biochemical abnormal stage by chip technology, and preliminarily explored the granulosa cells of lncRNAs in POI patients. Methods: 50 cases of POI patients were collected from September 2013 to December 2014 in the Chinese Han nationality biochemical abnormal stage. 50 cases of endocrine normal control women were treated with male and fallopian factors and 50 cases of granulosa cells were left. The difference table was screened in 10 cases of POI patients and 10 control female granulosa cells through the technique of POI chip. LncRNAs and mRNAs, according to the analysis results of Gene ontology and KEGG pathway on the expression spectrum chip, the co expression network analysis (co-expression networks) of the differentially expressed lncRNAs and mRNAs, according to the relative expression abundance, the multiplier and sequence information, is selected at the core position and is related to the important and differentially expressed genes. 14 lncRNAs were verified in 60 independent samples. Results: the results showed that 677.Real-Time PCR experiments of differential expression lncRNAs sequence showed that TCONS 00010009, TCONS 00014547, NR 40662.1, ENST00000520306, ENST00000420313, ENST00000526225 were significantly downregulated in POI patient granulosa cells. Bioinformatics analysis table All of them belong to lincRNAs, and there are many important coding genes around them. Especially, the DNA damage repair gene XRCC4 and MSH5 are located at the deficiency of 300KB near TCONS_00010009 and NR 040662, suggesting that these differentially expressed lincRNAs may participate in the POI disease by regulating the surrounding co expression genes. The initial study on the OncRNAs characteristics of granulosa cells by Arraystar Human IncRNA chip is the first time. There are many differentially expressed LncRNA in the granulosa cells of POI patients at the biochemical abnormal stage, which may be related to the disease.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R711.75

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