Mir-27a介导CRISP2表达导致弱畸形精子症的分子机制

发布时间：2018-05-29 02:58

本文选题：弱畸形精子症 + microRNA　；参考：《南方医科大学》2015年博士论文

【摘要】：全球范围内大概有15%的夫妻存在不孕不育,其中男性因素导致的不育占50%。在精液分析中,弱精症及弱畸形精子症是一个常见的精液异常指标；首先,我们采用人类全基因组基因芯片获取弱精症与正常男性精子的基因表达谱,通过生物信息学筛选差异表达基因,发现SEMG1基因可能是弱精症的候选基因之一；同时证实发现CRISP2在弱精子症中低表达,而且其他研究发现该蛋白为结构蛋白,在精卵结合的顶体反应及配子融合中其重要作用；其次,我们进一步评估CRISP2在弱畸形精子症精子中的表达及临床意义,发现CRISP2蛋白在弱畸形精子症中低表达,而Q-PCR结果无差异,提示在弱畸形精子症患者中,CRISP2蛋白的差异性表达是一种转录后的调控。因此,本向研究拟进一步评估该蛋白在弱畸形精子症低表达的分子机制。我们知道基因的异常表达常常因为遗传机制和表观遗传机制导致：表观遗传是指DNA序列不发生变化,但基因表达却发生了可遗传的改变。这种改变是细胞内除了遗传信息以外的其他可遗传物质发生的改变,且这种改变在发育和细胞增殖过程中能稳定传递。表观遗传分子机制包括DNA甲基化,组蛋白修饰,染色体改型及RNA干扰(MICRNA, LNCRNA)。我们对CRISP2基因的甲基化进行研究,采用甲基化特异性的PCR及亚硫酸盐序列测定法两种方法检测CRISP2的甲基化,在10例正常对照组和弱畸形精子症组中均未发现CRISP2基因启动子区CpG岛存在甲基化情况；CRISP2基因在20例正常精子及弱畸形精子症中精子中Q-PCR结果无差异,CRISP2蛋白表达显著下调。结合临床资料分析,发现CRISP2蛋白表达下调与精子形态、精子活力及男性不育呈正相关。CRISP2蛋白在弱畸形精子症患者中低表达,结合我们之前的CRISP2基因的甲基化的研究,未发现CRISP2基因启动子区CpG岛存在甲基化情况,表明CRISP2蛋白的表达受转录后的调控。进而我们将工作转向CRISP2基因/蛋白的靶向microRNA的研究。通过生物信息学软件miRDB、miRWalk、miRTargetscan预测作用于CRISP2的microRNA,选取评分最高、共同交集的microRNA来进一步研究：发现miR-27a、miR-27b、miR-340、miR-502-3p、miR-510、miR-640及miR-767-5p可以特异性结合CRISP2 3'-UTR区并调控其表达,有文献证实miR-27a在弱精症中高表达,因此,我们在20例正常精子及弱畸形精子症中精子中Q-PCR检测miR-27a表达,发现miR-27a在弱畸形精子症中精子中Q-PCR表达显著增加。结合临床资料分析,发现miR-27a表达下调与精子形态、精子活力及男性不育呈互相关。为此,我们进一步探讨是否miR-27a通过抑制CRISP2表达而导致弱畸形精子症的发生。在293细胞中进行荧光素酶报告基因Luciferase以验证筛选出的miR-27a和CRISP2基因的靶向性。通过将293细胞、含有CRISP2 3'-UTR(克隆的)pEZX-MT05 plasmid及miR-27a进行转染,取上清液进行蛋白荧光测定,测定结果提示miR-27a通过结合CRISP2 3'-UTR导致弱畸形精子症的发生可能。本研究的主要研究方法、内容和结论包括以下几个方面：第1部分基因芯片及生物信息学筛选精子活力不足相关差异表达基因目的：应用基因芯片技术获取成年男性精子活力不足和精子活力正常的精子基因表达谱,采用生物信息学筛选精子活力不足相关的差异表达基因,确定下一步研究目标。方法：1.收集成年男性弱精子症患者精液12例,活力正常精液12例作为对照组,Percoll密度梯度离心分离纯化后,提取精子总RNA,通过体外转录合成cRNA,逆转录标记cRNA,纯化后与Agilent 4110B芯片杂交,杂交信号经扫描后,Feature Extraction软件对扫描结果进行处理。2.将精子活力不足基因表达谱采用BRB-ArrayTools及GenSpring10.0分析工具进行分析,差异基因筛选采用Algorithm:GeneSpring10---t-Test Against Zero检验,检验结果由芯片公司提供。选取两种分析工具具有共同的交集的基因。3.我们采用如NextBio及MEDLINE和FACTA生物信息学软件工具对差异表达基因进一步筛选并通过荧光定量PCR对芯片检测结果进行验证。结果：1.将精子活力不足基因表达谱采用BRB-ArrayTools及GenSpring10.0分析工具进行分析,其中BRB-ArrayTools筛选得到的261个差异表达基因,而GenSpring10.0筛选得到的1265个差异表达基因,两种分析结果重叠的基因(表达上调或下调),即精子活力不足的分子标签,共有71个,其中10个基因共同表达上调,61个基因共同表达下调；2.我们在NextBio下载了畸精症和正常射精精子的基因芯片表达谱数据文件及其报道的差异基因,与我们分析得到的精子活力不足相关基因进行了进一步分析比较,发现21个在弱精症中特异表达的差异基因；3.进一步通过MEDLINE和FACTA软件工具分析我们发现PGAP1和SEMG1与男性不育有关,其中PGAP1与精子活力无关；4. Real-time PCR试验证实SEMG1基因的表达水平与芯片的结果基本一致。因此,我们提出SEMG1可能是精子活力不足的候选基因。结论：1.我们获得了精子活力不足的基因表达谱,芯片结果告诉我们精子活力与精子中的基因的表达水平可能有着密切的联系；2.筛选出的具有特征性的和新发现的基因可能会成为潜在的诊断的标记物；如生物信息学分析发现SEMG1可能是精子活力不足的候选基因；CRISP2基因在精子活力不足患者精子中表达显著下调,说明CRISP2与精子活力密切相关；这些基因的进一步功能研究可能会帮助我们解释精子活力不足的发病机制。第2部分CRISP2在弱畸形精子症中的表达及临床意义目的：探讨弱畸形精子症患者精子中富含半胱氨酸的分泌蛋白2(CRISP2)mRNA及蛋白的表达水平,明确其与弱畸形精子症关系,探讨其表达水平在临床中的意义。方法：1.收集成年男性弱畸形精子症患者精液20例,正常对照组精液20例作为对照组,50% Percoll离心分离纯化后,提取精子总RNA及总蛋白,采用SYBR Green实时定量PCR和免疫印迹(Western Blot)技术检测CRISP2 mRNA及蛋白相对表达量。2.通过回顾性调查弱畸形精子症及正常对照组1年,观察各组夫妻最终是否生育。采用Spearman相关系数(r)分析CRISP2 mRNA及蛋白相对表达量与弱畸形精子症及生育的相关性。所有数据采用GraphPad Prism 5软件进行统计分析。结果：1. CRISP2基因在弱畸形精子症患者精子中的表达量与其在正常对照组中的表达量间差异无统计学意义(P=-0.8578)。2. CRISP2蛋白在弱畸形精子症患者精子中的表达量与其在正常对照组中的表达量差异具有统计学意义(P=0.0156)。3. CRISP2蛋白表达量和精子前向运动率(r=0.6240,P=0.0011)及形态正常率(r=0.5845,P=0.0027)呈显著正相关;而CRISP2蛋白表达水平与年龄、精液量、pH值、精子浓度等无显著相关性。4.弱畸形精子症组中的不育率(73%)高于正常对照组(24%),且差异具有统计学意义(P=0.0188); CRISP2蛋白相对低表达组的不育率(67%)高于CRISP2蛋白相对高表达组的不育率(17%),差异具有统计学意义(P=0.0361)。结论：1. CRISP2蛋白表达水平与弱畸形精子症患者精子活力、形态呈显著正相关,而CRISP2蛋白低表达与弱畸形精子症患者生育预后呈正相关。2. CRISP2可能是弱畸形精子症发病机制研究的一个重要的分子靶标,值得进一步深入研究其作用及调控机制。第3部分Mir-27a在弱畸形精子症中的表达及临床意义目的：探讨弱畸形精子症患者精子中Mir-27a的表达水平,明确其与弱畸形精子症关系,探讨其表达水平在临床中的意义。方法：1.收集成年男性弱畸形精子症患者精液20例,正常对照组精液20例作为对照组,50%Percoll离心分离纯化后,提取精子总RNA,采用SYBR Green实时定量PCR检测Mir-27a RNA及蛋白相对表达量。3.通过回顾性调查弱畸形精子症及正常对照组1年,观察各组夫妻最终是否生育。采用相关性分析Mir-27a RNA表达量与弱畸形精子症及生育的相关性。所有数据采用GraphPad Prism 5软件进行统计分析。结果：1. Mir-27a在弱畸形精子症患者精子中的表达量与其在正常对照组中的表达量间具有统计学差异,表现为显著性高表达(P=0.0179)。2. Mir-27a基因和精子前向运动率(r=-0.3236,P=0.0417)及形态正常率(r=-0.4560,P=0.0031)呈显著负相关;而miR-27a表达水平与年龄、精液量、pH值、精子浓度等无显著相关性。3. Mir-27a基因相对低表达组的不育率(25%)显著低于miR-27a相对高表达组的不育率(69%,P=0.0320)。这些数据提示高水平的miR-27a与不育密切相关。结论：1. miR-27a基因表达水平与弱畸形精子症患者精子活力、形态呈显著负相关,而miR-27a高表达与弱畸形精子症患者生育预后呈负相关。2.提示miR-27a可能是弱畸形精子症发病机制研究的一个重要的分子靶标,值得进一步深入研究其作用及调控机制。第4部分Mir-27a介导CRISP2表达导致弱畸形精子症的分子机制目的：探讨弱畸形精子症患者精子中CRISP2表达下调的分子机制,明确Mir-27a是否与CRISP2有关,及其是否调控CRISP2的表达。方法：1.收集成年男性弱畸形精子症患者精液10例,正常对照组精液10例作为对照组,50%Percoll离心分离纯化后,提取精子DNA, PC扩增并利用BSP法直接扩增出硫化处理后的目的片段并测序,检测CRISP2基因的CpG岛甲基化情况。2.采用Spearman相关系数(r)分析弱畸形精子症中CRISP2基因／蛋白相对表达量与Mir-27a表达量的相关性。3.采用荧光素酶报告基因Luciferase验证miR-27a调控CRISP2,通过将293T细胞、含有CRISP2 3'-UTR(克隆的)pEZX-MT05 plasmid及miR-27a进行转染,取上清液进行蛋白荧光测定。所有数据采用GraphPad Prism 5软件进行统计分析。结果：1.10例正常组样本和10例弱畸形精症组样本CRISP2基因的CpG岛均未发生甲基化；2.弱畸形精子症组精液精子中miR-27a基因含量与CRISP2蛋白表达量呈显著负相关(r=-0.4330, P=0.0345); miR-27a基因含量与CRISP2基因表达量无相关(P0.05)；3.发现加入了miR-27a的含CRISP2基因3'UTR克隆组质粒的荧光活性显著低于其他组的活性,提示：miR-27a可以结合CRISP2基因3'UTR并抑制其表达。结论：1. CRISP2基因在弱畸形精子症及正常对照组中无表达差异,CRISP2基因在弱畸形精子症及正常对照组中无甲基化异常,而CRISP2蛋白在弱畸形精子症及正常对照组中表达存在显著差异,表达显著下调；2.通过生物信息学及Q-PCR验证发现miR-27a在弱畸形精子症及正常对照组中表达存在显著差异,表达显著上调,CRISP2蛋白与miR-27a两者之间存在显著负相关；3.进一步实验发现miR-27a对CRISP2蛋白的表达具有显著的抑制作用,这为下一步研究CRISP2及miR-27a的功能打下基础,为深入研究CRISP2信号通路提供了新的依据。
[Abstract]:About 15% of spouses worldwide have infertility, among which male infertility accounts for 50%. in semen analysis. Asthenospermia and asthenospermia is a common semen anomaly. First, we use human genome microarray to obtain gene expression profiles of asthenospermia and normal male sperm, through biology. Screening differentially expressed genes by informatics shows that SEMG1 gene may be one of the candidate genes for asthenospermia, and that CRISP2 is found to be low in asthenospermia, and other studies have found that the protein is structural protein, which is important in the acrosome reaction and gamete fusion of sperm oocyte binding. Secondly, we further evaluate CRISP2 in the presence of spermatozoa. The expression and clinical significance of spermatozoa in asthenospermia and the low expression of CRISP2 protein in asthenospermia, and no difference in Q-PCR results, suggesting that the differential expression of CRISP2 protein is a post transcriptional regulation in the patients with asthenospermia. Therefore, this study intends to further evaluate the low expression of the protein in the asthenospermia. The molecular mechanism. We know that the abnormal expression of genes is often caused by genetic and epigenetic mechanisms: epigenetics is that the DNA sequence does not change, but the gene expression has a genetic change. This change is a change in the genetic material other than the genetic information in the cell, and this change is The epigenetic molecular mechanisms include DNA methylation, histone modification, chromosome modification and RNA interference (MICRNA, LNCRNA) in the process of development and cell proliferation. We studied the methylation of the CRISP2 gene and detected the methylation of CRISP2 by two methods of methylation specific PCR and sulfite sequencing, at 10 The methylation of CpG island in the promoter region of CRISP2 gene was not found in the normal control group and the asthenospermia group. The Q-PCR results of the CRISP2 gene in the sperm of 20 normal spermatozoa and the asthenospermia were not different, and the expression of the CRISP2 protein was down significantly. The expression of CRISP2 protein and the morphology of the sperm were found by the analysis of the clinical data. The positive correlation between sperm motility and male infertility is a low expression of.CRISP2 protein in patients with asthenospermia. Combined with our previous methylation of the CRISP2 gene, the methylation of the CpG island of the CRISP2 gene promoter was not found, indicating that the expression of the CRISP2 protein was regulated after the transcriptional regulation. And then we turned the work to the CRISP2 gene. The study of protein targeting microRNA. Through the bioinformatics software miRDB, miRWalk, and miRTargetscan, it predicts the microRNA of CRISP2, and selects the highest grade and common intersection of microRNA to further study: miR-27a, miR-27b, miR-340, miR-502-3p, miR-510, etc. The expression of miR-27a was proved to be highly expressed in asthenospermia. Therefore, we detected the expression of miR-27a in the sperm of 20 normal spermatozoa and asthenospermia, and found that the expression of Q-PCR in the spermatozoa was significantly increased in the asthenospermia of the asthenospermia. The expression of miR-27a was significantly increased in the spermatozoa of the weak malformed spermatozoa. The expression of miR-27a and the morphology of sperm, the vitality of the sperm and the activity of sperm were found. Male infertility is interrelated. To this end, we further explore whether miR-27a leads to the occurrence of asthenospermia by inhibiting CRISP2 expression. The luciferase reporter gene Luciferase is used in 293 cells to verify the targeting of the selected miR-27a and CRISP2 genes. By 293 fine cells, CRISP2 3'-UTR (cloned) pEZX-MT05 pl Asmid and miR-27a were transfected and extracted from the supernatant for protein fluorescence. The results suggested that miR-27a could lead to the occurrence of asthenospermia by combining with CRISP2 3'-UTR. The main research methods, contents and conclusions of this study include the following aspects: first parts of gene core and bioinformatics screening sperm motility insufficiency The purpose of differentially expressed genes is to use gene chip technology to obtain the sperm gene expression profiles of adult male sperm motility insufficiency and normal sperm motility. The differential expression genes related to sperm motility are screened by bioinformatics to determine the next step of research. Methods: 1., 12 cases of semen of adult male asthenospermia were collected, and the vitality of the spermatozoa was collected. 12 normal semen were used as control group. After separation and purification of Percoll density gradient centrifugation, the total RNA of sperm was extracted, cRNA was transcribed in vitro and cRNA was marked by reverse transcription. After purification, hybridization with Agilent 4110B chip. After the hybridization signal was scanned, Feature Extraction software treated the scanned fruit by Feature Extraction software to extract the gene expression profile of sperm motility. BRB-ArrayTools and GenSpring10.0 analysis tools were used to analyze the differential gene screening using Algorithm:GeneSpring10---t-Test Against Zero test. The test results were provided by the chip company. We selected two analysis tools with a common intersection of gene.3.. We used NextBio and MEDLINE and FACTA bioinformatics software tools to do bad. The differentially expressed genes were further screened and tested by fluorescence quantitative PCR. Results: 1. the expression profiles of sperm motility insufficiency genes were analyzed by BRB-ArrayTools and GenSpring10.0 analysis tools, of which 261 differentially expressed genes were screened by BRB-ArrayTools, and 1265 differences were obtained by GenSpring10.0 screening. Different expression genes, two kinds of overlapping genes (up-regulated or down-regulated), that is, the molecular label of sperm motility insufficiency, there are 71, of which 10 genes are up regulated together, 61 genes are down regulated together; 2. we downloaded the gene chip expression profiles of abnormal spermatozoa and normal ejaculatory sperm in NextBio and reports The difference gene, which was further analyzed and compared with the gene of sperm motility insufficiency we analyzed, found 21 differentially expressed genes in asthenospermia. 3. further through MEDLINE and FACTA software tools, we found that PGAP1 and SEMG1 were associated with male infertility, and PGAP1 was not related to sperm motility; 4. Real-time PCR test verified that the expression level of the real SEMG1 gene is basically consistent with the results of the chip. Therefore, we suggest that SEMG1 may be a candidate gene for the deficiency of sperm motility. Conclusion: 1. we have obtained the gene expression profiles of insufficient sperm motility, and the results of the chip tell us that the sperm vitality may be closely linked to the level of the gene expression in the sperm. The characteristic and newly discovered genes screened by 2. may be a potential marker for diagnosis; for example, bioinformatics analysis found that SEMG1 may be a candidate gene for insufficient sperm motility, and the CRISP2 gene has a significant downregulation in sperm motility in patients with sperm motility, indicating that CRISP2 is closely related to sperm vitality; Further functional study of genes may help us explain the pathogenesis of insufficient sperm motility. Second the expression and clinical significance of CRISP2 in asthenospermia: To explore the expression level of cysteine rich protein 2 (CRISP2) mRNA and protein in spermatozoa of asthenospermia Methods: 1. to collect 20 cases of semen of adult male asthenospermia, 20 cases of normal control group and 20 cases of normal control group as control group. After 50% Percoll centrifugation, the total RNA and total protein of sperm were extracted. The real-time quantitative PCR and Western blot (Western Blot) technique of SYBR Green was used to detect the sperm. The relative expression of CRISP2 mRNA and protein.2. was examined by a retrospective survey of asthenospermia and normal control group for 1 years. The correlation between CRISP2 mRNA and relative expression of CRISP2 mRNA and protein relative expression was analyzed by Spearman correlation coefficient (R). All data were carried out by GraphPad Prism 5 software. Results: there was no significant difference between the expression of 1. CRISP2 gene in the sperm of the asthenospermia patients and the normal control group (P=-0.8578) the expression of.2. CRISP2 protein in the sperm of the asthenospermia patients was significantly different from that in the normal control group (P=0.0156) There was a significant positive correlation between the expression of.3. CRISP2 protein and the motility of sperm (r=0.6240, P=0.0011) and the normal rate of morphology (r=0.5845, P=0.0027), but there was no significant correlation between the expression level of CRISP2 protein and age, semen volume, pH, and sperm concentration in the.4. asthenospermia group (73%), which was higher than that of the normal control group (24%). Study significance (P=0.0188); the sterility rate of the relatively low expression group of CRISP2 protein (67%) was higher than that of the relatively high expression group of CRISP2 protein (17%), and the difference was statistically significant (P=0.0361). Conclusion: the expression level of 1. CRISP2 protein was positively correlated with the motility of spermatozoa in the patients with asthenospermia, and the low expression of CRISP2 protein and weak malformation. Positive correlation.2. CRISP2 may be an important molecular target for the study of the pathogenesis of asthenospermia. It is worth further studying its role and regulation mechanism. Third the expression and clinical significance of Mir-27a in asthenospermia, and its clinical significance: To explore the Mir-27 in spermatozoa of the weak malformed spermatozoa A expression level, clarify its relationship with asthenospermia and explore the significance of its expression level in clinical. Methods: 1. the semen of adult male asthenospermia was collected in 20 cases, and 20 cases of normal control group were used as control group. After 50%Percoll centrifugation was separated and purified, the total RNA of spermatozoa was extracted, and Mir-27a was used to detect Mir-27a by SYBR Green real-time quantitative PCR The relative expression of RNA and protein.3. was examined by a retrospective study of the asthenospermia and the normal control group for 1 years. The correlation between Mir-27a RNA expression and asthenospermia and fertility was analyzed by correlation analysis. All data were analyzed by GraphPad Prism 5 software. Results: 1. Mir-27a was weak. There was a statistical difference between the expression of sperm in the patients with malformed spermatozoa and the normal control group, which showed significant negative correlation between the significant high expression (P=0.0179).2. Mir-27a gene and the sperm forward movement rate (r=-0.3236, P=0.0417) and the normal rate of morphology (r=-0.4560, P= 0.0031); and the level of miR-27a expression and age, semen. There was no significant correlation between the amount, pH value, sperm concentration and so on. The sterility rate of the relatively low expression group of.3. Mir-27a (25%) was significantly lower than that of the relatively high expression group of miR-27a (69%, P=0.0320). These data suggest that the high level of miR-27a is closely related to infertility. Conclusion: the expression level of the 1. miR-27a gene and the sperm motility of the asthenospermia patients The high expression of miR-27a is negatively correlated with the prognosis of asthenospermia patients with negative correlation.2. suggesting that miR-27a may be an important molecular target for the study of the pathogenesis of asthenospermia. It is worth further studying its role and regulatory mechanism. The fourth part of Mir-27a mediates CRISP2 expression leading to asthenospermia. Molecular mechanism Objective: To investigate the molecular mechanism of down regulation of CRISP2 expression in sperm of asthenospermia patients, to determine whether Mir-27a is related to CRISP2 and to regulate the expression of CRISP2. Methods: 1. the semen of adult male asthenospermia was collected in 10 cases, and 10 cases of normal control group were used as control group, and 50%Percoll centrifugation was used for isolation and purification. Then, the sperm DNA, PC were amplified and the BSP method was used to directly amplify the target fragments after the vulcanization. The CpG island methylation of the CRISP2 gene was detected by Spearman correlation coefficient (R), and the correlation between the relative expression of the CRISP2 gene / protein and the expression of Mir-27a was analyzed by the Spearman correlation coefficient (R), and the luciferase reporter base was adopted. CRISP2 was regulated by miR-27a by Luciferase, and 293T cells were transfected with CRISP2 3'-UTR (cloned) pEZX-MT05 plasmid and miR-27a. The supernatant was used for the determination of protein fluorescence. All data were analyzed by GraphPad Prism 5 software. Results: 1.10 normal group samples and 10 cases of weak malformed spermatosis group were sampled. There was no methylation in the CpG islands, and the expression of miR-27a gene and CRISP2 protein in semen spermatozoa of 2. asthenozoospermia group were significantly different.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2

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