MicroRNAs在始基卵泡募集過程中的表達(dá)模式和調(diào)控機(jī)制的研究
發(fā)布時(shí)間：2019-01-27 06:22
【摘要】：[目的]篩選調(diào)節(jié)始基卵泡募集的]microRNAs(miRNAs)、探討miRNAs對(duì)始基卵泡募集的調(diào)控機(jī)制。 [方法]通過miRNA芯片技術(shù)檢測(cè)miRNAs在3天及5天小鼠卵巢中的表達(dá),實(shí)時(shí)定量PCR技術(shù)驗(yàn)證差異表達(dá)的miRNAs,利用NovelBio數(shù)據(jù)庫(kù)采用elim算法對(duì)差異表達(dá)的miRNAs進(jìn)行基因功能的顯著性分析。利用Fisher精確檢驗(yàn),得到P值,通過多重比較檢驗(yàn),確定pathway的FDR,最后得出顯著性信號(hào)通路。隨后基于GO及KEGG分析構(gòu)建miRNA-mRNA信號(hào)調(diào)節(jié)網(wǎng)絡(luò).采用原位雜交技術(shù)對(duì)有顯著意義的miR-145的表達(dá)進(jìn)行定量及定位。MiR-145antagomir處理體外培養(yǎng)3天乳鼠卵巢,共聚焦顯微鏡觀察antagomir進(jìn)入卵巢的情況,通過實(shí)時(shí)定量PCR技術(shù)驗(yàn)證其干擾效應(yīng)。HE染色、免疫組織化學(xué)、卵泡計(jì)數(shù)、PCR技術(shù)評(píng)估其對(duì)乳鼠卵泡發(fā)育的影響。采用PCR技術(shù)、免疫組織化學(xué)、Western Blot及熒光素酶活性檢測(cè)驗(yàn)證miR-145的靶基因。Western Blot檢測(cè)下調(diào)miR-145表達(dá)后對(duì)Smad、非Smad信號(hào)通路及凋亡相關(guān)蛋白表達(dá)的影響。 [結(jié)果】在5天乳鼠卵巢中顯著上調(diào)的miRNAs包括miR-122,miR-145,miR-380-3p,miR-680,miR-484,miR-496,miR-17,miR-1906,miR-136*,miR-125-3p等共15個(gè)。下調(diào)miRNAs包括miR-302*,miR-466g,miR-466-3p,miR-500,miR-382*,miR-574-3p,miR-1196等共9個(gè)。隨機(jī)挑選8個(gè)miRNAs進(jìn)行驗(yàn)證,實(shí)時(shí)定量PCR結(jié)果顯示差異表達(dá)的miRNAs表達(dá)趨勢(shì)同芯片結(jié)果一致,差異有顯著意義(P0.05),并證實(shí)miR-145為升高倍數(shù)排列第二的小分子RNA。生物信息學(xué)分析結(jié)果顯示,上調(diào)miRNA的靶基因參與的顯著功能包括細(xì)胞粘附、凋亡、小GTP酶介導(dǎo)的信號(hào)傳導(dǎo)、細(xì)胞周期、蛋白修飾過程、DNA修復(fù)、血管形成、離子轉(zhuǎn)運(yùn)等。下調(diào)miRNAs的靶基因參與的顯著功能則包括細(xì)胞粘附、細(xì)胞周期、凋亡、蛋白轉(zhuǎn)運(yùn)、細(xì)胞內(nèi)信號(hào)級(jí)聯(lián)傳遞、RNA剪切等等。上調(diào)]miRNA的靶基因參與的顯著信號(hào)通路49條,下調(diào)1miRNAs的靶基因參與顯著信號(hào)通路29條(P0.001)。在差異表達(dá)miRNAs參與的顯著信號(hào)通路中TGF-p信號(hào)通路在卵泡募集及發(fā)育過程中發(fā)揮重要作用,因此我們以參與TGF-p信號(hào)通路的所有有顯著意義的miRNAs及其參與顯著功能的靶基因?yàn)榛A(chǔ),構(gòu)建miRNA-mRNA調(diào)節(jié)網(wǎng)絡(luò)。由于在上調(diào)miRNAs中miR-145表達(dá)量排列第二,同時(shí)該miRNA調(diào)節(jié)的靶基因Tgfbr2、Acvr1b、Smad3、Smad5在TGF-p信號(hào)通路中均占據(jù)重要地位,故以miR-145為研究對(duì)象探討其調(diào)控機(jī)制。原位雜交結(jié)果顯示miR-145在3天中表達(dá)不明顯,在5天乳鼠卵巢初級(jí)卵泡中的顆粒細(xì)胞中表達(dá)明顯高于3天。MiR-145通過作用于靶基因Tgfbr2發(fā)揮效應(yīng),給予miR-145antagomir處理體外培養(yǎng)3天乳鼠卵巢,可使始基卵泡數(shù)量及初級(jí)卵泡絕對(duì)數(shù)量顯著減少(P0.05),但初級(jí)卵泡所占比例相對(duì)增加(P0.05)。進(jìn)一步研究發(fā)現(xiàn)MiR-145通過作用于靶基因Tgfbr2發(fā)揮效應(yīng)。MiR-145antagomir通過上調(diào)TGFBR2,激活Smad信號(hào)通路中的SMAD3而不是非Smad信號(hào)通路中的P38MAPK或JNK促進(jìn)始基卵泡細(xì)胞的凋亡。 [結(jié)論]在始基卵泡募集及始基卵泡維持的過程中,存在多個(gè)miRNAs的調(diào)控,miRNAs可通過調(diào)節(jié)多條信號(hào)通路發(fā)揮調(diào)節(jié)始基卵泡募集的過程。其中,miR-145在此過程中發(fā)揮重要作用,該小分子RNA通過調(diào)節(jié)TGFBR2的表達(dá)水平調(diào)節(jié)TGF-β信號(hào)通路進(jìn)而調(diào)控始基卵泡募集及維持始基卵泡池的功能。
[Abstract]:[Objective] To screen the microRNAs (miRNAs) for regulating the initial follicular recruitment, and to explore the regulation and control mechanism of miRNAs on the initial follicle recruitment.[Methods] The expression of miRNAs in the ovary of 3-and 5-day mice was detected by miRNA chip technology, and the differential expression of miRNAs was verified by real-time quantitative PCR. Analysis. Using Fisher's exact test, the P value was obtained, and the FDR of patway was determined by multiple comparative tests, and the significance signal was finally obtained. Construction of miRNA-mRNA signal regulatory network based on GO and KEGG analysis The expression of miR-145 in a significant sense is quantified and determined by in situ hybridization. position. MiR-145antagomir treated in vitro for 3 days of rat ovarian, confocal microscope to observe the condition of anagomir into the ovary, and the interference effect was verified by real-time quantitative PCR. The results of HE staining, immunohistochemistry, follicular count, and PCR were used to assess the development of the follicle in the rat. in response to that detection of the target group of miR-145 using PCR technique, immunohistochemistry, Western Blot and luciferase activity detection The expression of Smad, non-Smad signaling pathway and apoptosis-related protein after down-regulation of miR-145 expression by Western Blot detection In response.[Results] miRNAs that were significantly up-regulated in the 5-day rat ovary include miR-122, miR-145, miR-380-3p, miR-680, miR-484, miR-496, miR-17, miR-1906, miR-136 *, miR-125-3p, and the like. 15. The down-regulation of miRNAs includes miR-302 *, miR-466g, miR-466-3p, miR-500, miR-382 *, miR-574-3p, miR-1196, and the like. 9. 8 miRNAs were randomly selected for validation, and the real-time quantitative PCR results showed that the expression of miRNAs in the differential expression was consistent with the results of the chip, and the difference was significant (P0.05), and it was confirmed that the miR-145 was the second small molecule in the increasing multiple. The results of bioinformatics analysis show that the significant functions of up-regulation of the target gene of miRNAs include cell adhesion, apoptosis, small GTP-mediated signaling, cell cycle, protein modification, DNA repair, angiogenesis, ion, The significant function of down-regulation of the target gene of miRNAs includes cell adhesion, cell cycle, apoptosis, protein transport, intracellular signal cascade transfer, RNA scissors, The target gene of the miRNAs was adjusted to participate in the significant signaling pathway 49, and the target gene for down-regulation of the 1miRNAs was involved in the significant signaling pathway 29 (P0.001). 01) The TGF-p signaling pathway plays an important role in the recruitment and development of the follicle in the significant signaling pathway involved in the differential expression of miRNAs, so we build miRNA-mRNA tones on the basis of all the significant miRNAs involved in the TGF-p signaling pathway and their target genes involved in the significant function Because the expression of miR-145 in the up-regulated miRNAs is the second, and the target genes Tgfbr2, Acvr1b, Smad3 and Smad5, which are regulated by the miRNA, occupy an important position in the TGF-p signal path, the miR-145 is used as the research object to discuss the modulation. The results of in situ hybridization show that the expression of miR-145 is not obvious in 3 days, and the expression of miR-145 in the granulosa cells in the primary follicle of the ovary of the 5-day milk rat is obviously high. The effect of MiR-145 on the target gene Tgfbr2, and the effect of the MiR-145 on the target gene Tgfbr2, and the administration of the miR-145antagomir to the in vitro culture for 3 days of the ovary of the rat, the number of the initial follicle and the absolute number of the primary follicle can be significantly reduced (P0.05), but the proportion of the primary follicle is relatively increased (P0. 05). Further study found that MiR-145 was produced by acting on the target gene Tgfbr2 The swing effect. MiR-145antagomir, by up-regulating the TGFBR2, activates the SMAD3 in the Smad signal path instead of the P38MAPK or JNK in the non-Smad signal path to promote the start-up follicle cells[Conclusion] There are multiple miRNAs in the process of the initial follicle recruitment and the maintenance of the initial follicle, and the miRNAs can be used to regulate the starting-base follicle by regulating the multiple signal pathways. In the process of raising, the miR-145 plays an important role in the process, and the small-molecule RNA regulates the TGF-I signal path through the regulation of the expression level of the TGFBR2, so as to control the starting-base follicle to raise and maintain the initial-based egg.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321
本文編號(hào)：2416002
[Abstract]:[Objective] To screen the microRNAs (miRNAs) for regulating the initial follicular recruitment, and to explore the regulation and control mechanism of miRNAs on the initial follicle recruitment.[Methods] The expression of miRNAs in the ovary of 3-and 5-day mice was detected by miRNA chip technology, and the differential expression of miRNAs was verified by real-time quantitative PCR. Analysis. Using Fisher's exact test, the P value was obtained, and the FDR of patway was determined by multiple comparative tests, and the significance signal was finally obtained. Construction of miRNA-mRNA signal regulatory network based on GO and KEGG analysis The expression of miR-145 in a significant sense is quantified and determined by in situ hybridization. position. MiR-145antagomir treated in vitro for 3 days of rat ovarian, confocal microscope to observe the condition of anagomir into the ovary, and the interference effect was verified by real-time quantitative PCR. The results of HE staining, immunohistochemistry, follicular count, and PCR were used to assess the development of the follicle in the rat. in response to that detection of the target group of miR-145 using PCR technique, immunohistochemistry, Western Blot and luciferase activity detection The expression of Smad, non-Smad signaling pathway and apoptosis-related protein after down-regulation of miR-145 expression by Western Blot detection In response.[Results] miRNAs that were significantly up-regulated in the 5-day rat ovary include miR-122, miR-145, miR-380-3p, miR-680, miR-484, miR-496, miR-17, miR-1906, miR-136 *, miR-125-3p, and the like. 15. The down-regulation of miRNAs includes miR-302 *, miR-466g, miR-466-3p, miR-500, miR-382 *, miR-574-3p, miR-1196, and the like. 9. 8 miRNAs were randomly selected for validation, and the real-time quantitative PCR results showed that the expression of miRNAs in the differential expression was consistent with the results of the chip, and the difference was significant (P0.05), and it was confirmed that the miR-145 was the second small molecule in the increasing multiple. The results of bioinformatics analysis show that the significant functions of up-regulation of the target gene of miRNAs include cell adhesion, apoptosis, small GTP-mediated signaling, cell cycle, protein modification, DNA repair, angiogenesis, ion, The significant function of down-regulation of the target gene of miRNAs includes cell adhesion, cell cycle, apoptosis, protein transport, intracellular signal cascade transfer, RNA scissors, The target gene of the miRNAs was adjusted to participate in the significant signaling pathway 49, and the target gene for down-regulation of the 1miRNAs was involved in the significant signaling pathway 29 (P0.001). 01) The TGF-p signaling pathway plays an important role in the recruitment and development of the follicle in the significant signaling pathway involved in the differential expression of miRNAs, so we build miRNA-mRNA tones on the basis of all the significant miRNAs involved in the TGF-p signaling pathway and their target genes involved in the significant function Because the expression of miR-145 in the up-regulated miRNAs is the second, and the target genes Tgfbr2, Acvr1b, Smad3 and Smad5, which are regulated by the miRNA, occupy an important position in the TGF-p signal path, the miR-145 is used as the research object to discuss the modulation. The results of in situ hybridization show that the expression of miR-145 is not obvious in 3 days, and the expression of miR-145 in the granulosa cells in the primary follicle of the ovary of the 5-day milk rat is obviously high. The effect of MiR-145 on the target gene Tgfbr2, and the effect of the MiR-145 on the target gene Tgfbr2, and the administration of the miR-145antagomir to the in vitro culture for 3 days of the ovary of the rat, the number of the initial follicle and the absolute number of the primary follicle can be significantly reduced (P0.05), but the proportion of the primary follicle is relatively increased (P0. 05). Further study found that MiR-145 was produced by acting on the target gene Tgfbr2 The swing effect. MiR-145antagomir, by up-regulating the TGFBR2, activates the SMAD3 in the Smad signal path instead of the P38MAPK or JNK in the non-Smad signal path to promote the start-up follicle cells[Conclusion] There are multiple miRNAs in the process of the initial follicle recruitment and the maintenance of the initial follicle, and the miRNAs can be used to regulate the starting-base follicle by regulating the multiple signal pathways. In the process of raising, the miR-145 plays an important role in the process, and the small-molecule RNA regulates the TGF-I signal path through the regulation of the expression level of the TGFBR2, so as to control the starting-base follicle to raise and maintain the initial-based egg.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321
【參考文獻(xiàn)】
中國(guó)期刊全文數(shù)據(jù)庫(kù) 前1條
1 周瑩;朱英哲;張素華;王紅梅;王樹玉;楊曉葵;;卵巢早衰microRNA的差異表達(dá)及其作用[J];中國(guó)優(yōu)生與遺傳雜志;2011年05期
，本文編號(hào)：2416002
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