miRNA-133b调控卵泡发育的分子机制

发布时间：2018-06-13 03:40

本文选题：MicroRNA + 卵泡发育　；参考：《南华大学》2014年博士论文

【摘要】：国家卫生和计划生育委员会最新数据表明,2013年我国不孕症发病率7%~10%,并有逐年上升的趋势。在不孕症的诸多病因中,广泛排卵障碍性不孕是女性不孕症常见的类型,约占25%~35%[1-3]。卵泡发育成熟障碍可引起不排卵,进而导致不孕,研究卵泡发育成熟障碍的调控机制是目前生殖领域的热点。卵泡发育是一个极其复杂的过程,包括不同时期卵泡形态和功能的变化,受内分泌、局部因子、基因和微小RNA(miRNA)等多因素调控。对卵泡发育调控机制已经取得一些进展,但还有很多问题亟待解决。本研究用miRNA基因芯片筛选在模拟人体卵巢内环境的条件下,不同发育时期的人卵母细胞差异表达的mi RNA,选取特异性高表达miRNA作为研究靶标,用生物信息学方法分析靶标miRNA的靶基因;通过研究靶标miRNA对靶基因表达及功能的影响,探讨靶标miRNA调控卵细胞发育成熟的分子机制,寻找调控卵细胞发育成熟的药物靶点,为防治不孕不育提供提供新的实验依据。第一部分不同发育期人卵母细胞miRNA的差异表达目的:筛选在模拟人体卵巢内环境的条件下,不同发育时期的人卵母细胞差异表达的miRNA,选取特异性高表达miRNA作为研究靶标,分析靶标miRNA的靶基因。方法:收集人GV期、MI期卵母细胞,用含胰岛素样生长因子-1(IGF-1)的培养液培养,以模拟人卵巢内环境,用miRNA微阵列技术筛选不同发育时期的卵母细胞差异性表达的miRNAs;用生物信息学技术预测其靶基因。结果:用含IGF-1的培养液培养,GV期卵母细胞183个miRNA表达上调,117个mi RNA表达下调;MI期卵母细胞145个miRNA表达上调,200个miRNA表达下调。其中,miR-133b在MI期卵母细胞的表达上调32倍,在GV期卵母细胞miR-133b表达无差异。靶基因分析软件显示miR-133b的一个靶基因是TAGLN2;miR-133b的结合位点在TAGLN2 3’UTR第215-250位核苷酸。小结:在模拟人体卵巢内环境的条件下,GV期卵母细胞183个mi RNA表达上调,117个miRNA表达下调,MI期卵母细胞145个miRNA表达上调,200个miRNA表达下调。mi R-133b是MI期卵母细胞特异性高表达的miRNA,其靶基因可能是TAGLN2。第二部分mi R-133b与TAGLN2基因的相互作用目的:分析mi R-133b与转胶蛋白2(TAGLN2)的相互作用,确证TAGLN2是mi R-133b的靶基因。方法:用基因重组技术构建TAGLN2-3’UTR及其突变表达载体(分别命名为psi CHECK-TAGLN2-3’UTR和psi CHECK-TAGLN2-3’UTR-m),用mi R-133b分别与psi CHECK或psi CHECK-TAGLN2-3’UTR或psi CHECK-TAGLN2-3’UTR-m共转染HEK 293T,用双荧光素酶报告基因检测系统分析mi R-133b与TAGLN2结合情况,用免疫印迹和Realtime-PCR分析mi R-133b对TAGLN2表达的影响,免疫荧光分析TAGLN2在MI期卵母细胞的表达及其在亚细胞定位。结果:成功构建psi CHECK-TAGLN2-3’UTR和psi CHECK-TAGLN2-3’UTR-m表达载体,mi R-133b与psi CHECK-TAGLN2-3’UTR共转染HEK 293T细胞后,荧光素酶活性显著降低(p=0.000.01),mi R-133b与空载体psi CHECK共转染HEK 293T细胞后,荧光素酶活性没有明显变化(p=0.590.05)。mi R-133b与psi CHECK-TAGLN2-3’UTR-m共转染HEK 293T细胞后,荧光素酶活性也没有明显变化(p=0.620.05)。mi R-133b与TAGLN2 3’UTR第215-250位核苷酸序列结合。mi R-133b mimics使TAGLN2表达下调;mi R-133b inhibitor可以使TAGLN2表达上调。TAGLN2定位分布人MI期卵母细胞浆,细胞核较少。结论:mi R-133b是MI卵母细胞特异性高表达mi RNA,其靶基因是TAGLN2、结合位点是TAGLN2的3’UTR第215-250位核苷酸序列。mi R-133b下调TAGLN2 m RNA和蛋白的表达。TAGLN2定位于人MI期卵母细胞浆。第三部分mi R-133b靶向TAGLN2调控卵泡的发育成熟目的:探讨mi R-133b靶向TAGLN2调控卵泡发育成熟的机制。方法:首先,选择4周龄和8周龄的雌性ICR小鼠,各6只,采用免疫印记方法检测TAGLN2在4周龄和8周龄小鼠卵巢的表达水平。第二,随机选择4周龄雌性ICR小鼠,分4组,每组6只,采用免疫组化方法检测TAGLN2在窦前卵泡、窦状卵泡、排卵前卵泡及黄体中的定位和表达。第三,随机选择8周龄雌性ICR小鼠,分为3组,每组10只,用免疫荧光染色方法检测GV期、GVBD期及MII期卵母细胞中TAGLN2的表达水平。第四,随机选择8周龄雌性ICR小鼠,分成3组,每组8只,采用TAGLN2 si RNA分析沉默TAGLN2基因前后,卵母细胞直径、透明带厚度变化情况。第五,随机选择8周龄雌性ICR小鼠,随机分成5组,每组8只,通过卵巢多点注射法,将mi R-133b模拟物(mi R-133b minic)、mi R-133b模拟物的阴性对照(mi R-133b minic negative control)、mi R-133b抑制物(mi R-133b inhibitor)、mi R-133b抑制物的阴性对照(mi R-133b inhibitor negative control)及上述制剂的溶剂注射入卵巢,分别统计每只小鼠获卵数,用免疫印迹和实时定量PCR方法检测卵巢TAGLN2的表达。第六,收集GV期卵母细胞,随机分成5组通过显微注射法将mi R-133b minic、mi R-133b minic negative control、mi R-133b inhibitor、mi R-133b inhibitor negative control及上述制剂的溶剂注射至卵母细胞胞浆,观察各组卵母细胞的成熟情况。分析mi R-133b对小鼠卵母细胞成熟的影响。结果:免疫印记显示TAGLN2在4周龄和8周龄小鼠卵巢组织均有表达,8周龄小鼠卵巢TAGLN2的表达水平显著高于4周龄小鼠卵巢(p=0.0320.05),免疫组化显示TAGLN2在窦前卵泡、窦状卵泡、排卵前卵泡、黄体有表达,且定位于颗粒细胞包膜以及胞浆,细胞核几乎无表达。颗粒细胞TAGLN2的表达水平随着卵泡发育逐渐降低,排卵前卵泡达到最低(p=0.0060.01p0.05),至黄体期其表达水平较排卵前卵泡增强。免疫荧光染色显示TAGLN2在小鼠卵母细胞胞浆表达,GVBD期卵母细胞TAGLN2的表达水平显著低于GV期卵母细胞的表达水平(p=0.000.01),MⅡ期卵泡卵母细胞中TAGLN2的表达水平最低(p=0.020.05)。与对照组相比,TAGLN2-si RNA处理组卵母细胞直径显著增大(p=0.010.05),透明带厚度变化无显著差异(p0.05)。分别给小鼠卵巢及卵母细胞注射mi R-133b minic、mi R-133b minic negative control、mi R-133b inhibitor、mi R-133b inhibitor negative control以注射上述制剂的溶剂作为对照,结果显示:与对照组比较,mi R-133b mimic处理组GV期和MII期卵母细胞数增多,但无统计学意义(p0.05),mi R-133b inhibitor处理组小鼠GV期和MII期卵母细胞数显著降低(p0.05);mi R-133b mimic显著诱导卵母细胞的成熟(p0.05),mi R-133b inhibitor显著抑制卵母细胞的成熟(p0.05)。mi R-133b mimic下调TAGLN2的表达,mi R-133b inhibitor上调TAGLN2的表达。结论TAGLN2定位于卵泡中颗粒细胞膜和细胞质,其表达与卵母细胞成熟呈负相关。mi R-133b抑制小鼠卵巢TAGLN2 m RNA及蛋白的表达,促进小鼠卵母细胞成熟。
[Abstract]:The latest data from the National Committee for health and family planning shows that the incidence of infertility in China is 7%~10% in 2013 and has been increasing year by year. Among the many causes of infertility, extensive ovulatory infertility is a common type of female infertility, which accounts for the development of 25%~35%[1-3]. follicles to cause infertile, and then lead to infertility. The regulation mechanism of follicle development disorder is a hot spot in the field of reproductive development. Follicle development is an extremely complex process, including changes in follicle morphology and function at different times, regulated by many factors such as endocrine, local factor, gene and small RNA (miRNA). Many problems need to be solved. This study uses miRNA gene chip to screen mi RNA differentially expressed in human oocytes at different developmental stages, select specific high expression miRNA as research target and analyze target gene of target miRNA by bioinformatics, and study target gene table by target miRNA. To explore the molecular mechanism of the target miRNA regulating the maturation of oocyte, to find the drug targets to regulate the maturation of oocyte, and to provide new experimental basis for the prevention and control of infertility. The first part of the differential expression of miRNA in human oocyte at different developmental stages: screening the conditions in the simulation of human ovarian environment. The differential expression of miRNA in human oocytes at different developmental stages selected specific high expression miRNA as the research target and analyzed target gene of target miRNA. Methods: to collect human GV, MI oocytes, culture medium containing insulin-like growth factor -1 (IGF-1), to simulate the human ovarian environment, and to screen by miRNA microarray technology. The differentially expressed miRNAs in the oocytes of the same developmental period; the target gene was predicted by bioinformatics. Results: the expression of 183 miRNA expressions in GV oocytes was up-regulated by IGF-1 culture medium, and the expression of 117 mi RNA was down regulated, the expression of 145 miRNA in MI oocytes was up and 200 miRNA expressions downregulated. Among them, miR-133b was in MI phase oocyte. The expression of the cell was up to 32 times, and there was no difference in the expression of miR-133b in GV oocyte. The target gene analysis software showed that a target gene of miR-133b was TAGLN2, and the binding site of miR-133b was in TAGLN2 3 'UTR 215-250 nucleotide. Down regulation, the expression of 145 miRNA in MI oocytes was up, and 200 miRNA expression down regulated.Mi R-133b was the specific and high expression of miRNA in MI oocytes. The target gene may be the interaction between TAGLN2. second mi R-133b and TAGLN2 gene. Methods: TAGLN2-3 'UTR and its mutant expression vectors were constructed by gene recombination technology (named psi CHECK-TAGLN2-3' UTR and psi CHECK-TAGLN2-3 'UTR-m, respectively). Analysis of the combination of MI R-133b with TAGLN2, the effects of MI R-133b on the expression of TAGLN2 by immunoblotting and Realtime-PCR, and the expression of TAGLN2 in MI oocyte and its subcellular location by immunofluorescence. After N2-3 'UTR co transfected HEK 293T cells, luciferase activity decreased significantly (p=0.000.01). After MI R-133b and psi CHECK co transfected HEK 293T cells, luciferase activity was not significantly changed. 05).Mi R-133b and TAGLN2 3 'UTR nucleotide sequence combined with.Mi R-133b mimics to reduce the expression of TAGLN2, MI R-133b inhibitor can make TAGLN2 expression up-regulated and distributes human oocyte pulp with fewer nuclei. The point is TAGLN2 3 'UTR nucleotide sequence 215-250 nucleotide sequence.Mi R-133b downregulates TAGLN2 m RNA and protein expression.TAGLN2 located in human MI oocyte pulp. Third mi R-133b targeting TAGLN2 regulates the maturation of follicles. The expression level of TAGLN2 in the ovary of 4 week and 8 week old mice was detected by immuno imprinting. Second, 4 weeks old female ICR mice were randomly selected, and 6 rats in each group were divided into 4 groups. The location and expression of TAGLN2 in preantral follicles, antral follicles, preovulal follicles and corpus luteum were detected by immunohistochemistry. Third, random. 8 weeks old female ICR mice were selected to be divided into 3 groups, 10 rats in each group. The expression level of TAGLN2 in phase GV, GVBD and MII oocytes was detected by immunofluorescence. Fourth, 8 weeks old female ICR mice were randomly selected and divided into 3 groups, 8 in each group. The diameter of oocyte and the thickness of the zona pellucida were changed by TAGLN2 Si RNA analysis. Fifth, randomly selected 8 weeks old female ICR mice, randomly divided into 5 groups, 8 rats in each group. The MI R-133b analogue (MI R-133b minic), the negative control of MI R-133b analog (MI R-133b minic negative), and the negative control inhibitor of MI R-133b, were randomly assigned to each group. Ibitor negative control) and the solvent of the above preparation were injected into the ovary. The number of eggs obtained in each mouse was measured respectively. The expression of TAGLN2 in the ovary was detected by immunoblotting and real-time quantitative PCR. Sixth, the GV oocyte was collected, and the MI R-133b minic was randomly divided into 5 groups by microinjection, MI R-133b minic. Bitor, MI R-133b inhibitor negative control and the solvent of the aforementioned preparation were injected into the oocyte cytoplasm to observe the maturation of oocyte in each group. The effect of MI R-133b on the maturation of oocyte in mice was analyzed. Results: the immune imprint showed that TAGLN2 was expressed at 4 weeks and 8 weeks of age in mouse ovary tissues, and the table of TAGLN2 in the ovary of 8 weeks old mice The level was significantly higher than the 4 week old mouse ovary (p=0.0320.05). The immunohistochemical staining showed that TAGLN2 was in preantral follicles, sinus like follicles, pre ovulation follicles, and the corpus luteum was expressed in the granulosa cell envelope and cytoplasm, and the nucleus was almost no expression. The expression of TAGLN2 in granular cells decreased gradually with the follicle development, and the follicles reached the lowest level before ovulation. The expression level of p=0.0060.01p0.05 in the luteal phase was stronger than that before ovulation. Immunofluorescence staining showed that TAGLN2 was expressed in the cytoplasm of mouse oocyte, and the expression level of TAGLN2 in GVBD oocyte was significantly lower than that of GV oocyte (p=0.000.01), and the expression level of TAGLN2 in M II oocyte was the lowest (p=0.020.05). Compared with the control group, the oocyte diameter of the TAGLN2-si RNA treatment group increased significantly (p=0.010.05), and there was no significant difference in the thickness of the zona pellucida (P0.05). The mice ovary and oocyte were injected with MI R-133b minic, and MI R-133b minic negative control was injected. As compared with the control group, the results showed that compared with the control group, the number of GV and MII oocytes increased in MI R-133b mimic treatment group, but there was no statistical significance (P0.05). The number of oocytes in GV and MII phase of MI R-133b inhibitor treated mice decreased significantly (P0.05). The maturation of oocyte (P0.05).Mi R-133b mimic down regulated the expression of TAGLN2, and MI R-133b inhibitor up-regulated the expression of TAGLN2. Conclusion TAGLN2 is located in the membrane and cytoplasm of granulosa cells in the follicle, and the expression is negatively correlated with the maturation of oocyte. The cell matures.
【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.6

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