MicroRNAs在始基卵泡募集过程中的表达模式和调控机制的研究
发布时间：2019-01-27 06:22
【摘要】：[目的]筛选调节始基卵泡募集的]microRNAs(miRNAs)、探讨miRNAs对始基卵泡募集的调控机制。 [方法]通过miRNA芯片技术检测miRNAs在3天及5天小鼠卵巢中的表达,实时定量PCR技术验证差异表达的miRNAs,利用NovelBio数据库采用elim算法对差异表达的miRNAs进行基因功能的显著性分析。利用Fisher精确检验,得到P值,通过多重比较检验,确定pathway的FDR,最后得出显著性信号通路。随后基于GO及KEGG分析构建miRNA-mRNA信号调节网络.采用原位杂交技术对有显著意义的miR-145的表达进行定量及定位。MiR-145antagomir处理体外培养3天乳鼠卵巢,共聚焦显微镜观察antagomir进入卵巢的情况,通过实时定量PCR技术验证其干扰效应。HE染色、免疫组织化学、卵泡计数、PCR技术评估其对乳鼠卵泡发育的影响。采用PCR技术、免疫组织化学、Western Blot及荧光素酶活性检测验证miR-145的靶基因。Western Blot检测下调miR-145表达后对Smad、非Smad信号通路及凋亡相关蛋白表达的影响。 [结果】在5天乳鼠卵巢中显著上调的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个。下调miRNAs包括miR-302*,miR-466g,miR-466-3p,miR-500,miR-382*,miR-574-3p,miR-1196等共9个。随机挑选8个miRNAs进行验证,实时定量PCR结果显示差异表达的miRNAs表达趋势同芯片结果一致,差异有显著意义(P0.05),并证实miR-145为升高倍数排列第二的小分子RNA。生物信息学分析结果显示,上调miRNA的靶基因参与的显著功能包括细胞粘附、凋亡、小GTP酶介导的信号传导、细胞周期、蛋白修饰过程、DNA修复、血管形成、离子转运等。下调miRNAs的靶基因参与的显著功能则包括细胞粘附、细胞周期、凋亡、蛋白转运、细胞内信号级联传递、RNA剪切等等。上调]miRNA的靶基因参与的显著信号通路49条,下调1miRNAs的靶基因参与显著信号通路29条(P0.001)。在差异表达miRNAs参与的显著信号通路中TGF-p信号通路在卵泡募集及发育过程中发挥重要作用,因此我们以参与TGF-p信号通路的所有有显著意义的miRNAs及其参与显著功能的靶基因为基础,构建miRNA-mRNA调节网络。由于在上调miRNAs中miR-145表达量排列第二,同时该miRNA调节的靶基因Tgfbr2、Acvr1b、Smad3、Smad5在TGF-p信号通路中均占据重要地位,故以miR-145为研究对象探讨其调控机制。原位杂交结果显示miR-145在3天中表达不明显,在5天乳鼠卵巢初级卵泡中的颗粒细胞中表达明显高于3天。MiR-145通过作用于靶基因Tgfbr2发挥效应,给予miR-145antagomir处理体外培养3天乳鼠卵巢,可使始基卵泡数量及初级卵泡绝对数量显著减少(P0.05),但初级卵泡所占比例相对增加(P0.05)。进一步研究发现MiR-145通过作用于靶基因Tgfbr2发挥效应。MiR-145antagomir通过上调TGFBR2,激活Smad信号通路中的SMAD3而不是非Smad信号通路中的P38MAPK或JNK促进始基卵泡细胞的凋亡。 [结论]在始基卵泡募集及始基卵泡维持的过程中,存在多个miRNAs的调控,miRNAs可通过调节多条信号通路发挥调节始基卵泡募集的过程。其中,miR-145在此过程中发挥重要作用,该小分子RNA通过调节TGFBR2的表达水平调节TGF-β信号通路进而调控始基卵泡募集及维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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321
本文编号：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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321
【参考文献】
中国期刊全文数据库 前1条
1 周莹;朱英哲;张素华;王红梅;王树玉;杨晓葵;;卵巢早衰microRNA的差异表达及其作用[J];中国优生与遗传杂志;2011年05期
，本文编号：2416002
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