多囊卵巢综合征大鼠模型卵巢中microRNAs表达及其相关功能的研究

发布时间：2018-04-26 03:07

本文选题：多囊卵巢综合征 + microRNA　；参考：《吉林大学》2016年博士论文

【摘要】：多囊卵巢综合征(polycystic ovary syndrome,PCOS)是一种复杂的、异质性内分泌紊乱综合征,以高雄激素血症、高胰岛素血症、胰岛素抵抗和持续性无排卵为主要特征。PCOS患者的临床表现多样,其近期和远期并发症对女性的身心健康影响较大。据报道,PCOS是无排卵性不孕的最常见原因,另外PCOS患者2型糖尿病、心血管疾病和子宫内膜癌的发病风险与非PCOS患者相比明显升高。鉴于PCOS对女性健康的巨大危害,对其发病机制的研究已经成为全球妇科内分泌专家关注的焦点。目前认为PCOS可能的致病原因包括雄激素生成过多、下丘脑垂体功能紊乱、胰岛素抵抗、细胞凋亡及卵泡发育异常等,其中细胞凋亡和卵泡发育异常导致PCOS发病的理论受到越来越多学者的关注,但是具体的发病机制尚需要进一步挖掘。微小RNA(micro RNA,mi RNA)是一类内源性单链、非编码的小分子RNA,一般长度为18-24核苷酸,通过与其靶基因3’非编码区(untranslated region,UTR)结合,在转录后水平负性调控其靶基因。近年来,人们发现mi RNA在子宫、输卵管、卵巢等女性生殖器官中均有表达,并广泛参与调控女性卵泡发育成熟、受精、着床及胚胎发育等生理过程,对维持女性正常生育功能起重要作用。同时研究还发现,mi RNA的异常表达可能与很多女性疾病的发生有关,如PCOS患者卵泡颗粒细胞中的mi RNA存在异常表达,而其中一部分mi RNAs(如let-7a、let-7i和mi R-92b)的异常表达可能与卵泡颗粒细胞凋亡有关。由此可见,mi RNA可能通过影响颗粒细胞凋亡,进而导致PCOS的发生。本研究的目的在于揭示对PCOS发病起关键作用的mi RNA及其导致疾病发生的调控作用机制。由于PCOS患者颗粒细胞取材困难,因此本研究构建了PCOS大鼠模型,借助mi RNA深度测序、荧光定量PCR、Western Blot、基因转染和双荧光素酶报告基因检测等实验技术,挖掘可能与PCOS疾病发生相关的mi RNAs及其下游调控通路。主要研究内容如下:1.PCOS大鼠模型的构建我们采用来曲唑灌胃法建立PCOS大鼠模型。PCOS模型组:每日将来曲唑按1mg/(kg?d)溶解到1%羧甲基纤维素(CMC)中,连续23天灌胃处理;对照组:每日CMC 1mg/(kg?d),连续23天灌胃处理。结果:来曲唑灌胃处理后,大鼠阴道涂片显示,对照组大鼠存在规律的动情周期变化,而模型组大鼠的动情周期失去规律性变化,模型组大鼠的阴道涂片可见大量白细胞,提示大鼠持续处于动情间期。卵巢组织HE染色显示模型组卵巢结构紊乱,卵巢内囊状扩张的卵泡增多、黄体及发育阶段卵泡数目明显减少。ELISA检测显示,模型组大鼠血清LH、FSH、T浓度显著高于对照组,E2浓度显著低于对照组。以上实验数据说明我们成功构建了PCOS大鼠模型。2.深度测序及生物信息学分析提取PCOS模型组与对照组大鼠卵巢组织的总RNA,经深度测序后发现,显著差异表达的mi RNAs共有129个,其中表达上调的mi RNAs有49个,表达下调的mi RNAs有80个。选取4个与细胞增殖、凋亡相关的mi RNAs进行验证,荧光定量PCR结果显示,mi R-34b-5p、mi R-141-3p和mi R-200a-3p在模型组大鼠卵巢组织中呈显著性下调,而mi R-201-5p在模型组大鼠卵巢组织中呈显著性上调,进一步验证了深度测序结果的准确性。随后利用生物信息学软件对4个差异表达mi RNAs进行下游靶基因预测,共发现2060个靶基因。运用Gene Ontology(GO)、Pathway analysis等软件对这些靶基因的潜在下游调控通路进行富集和筛选,发现4个差异表达mi RNAs的靶基因参与卵母细胞减数分裂、MAPK信号通路、PI3K-Akt信号通路、Rap1信号通路及Notch信号通路、生殖过程和细胞凋亡等多条信号通路。3.选取mi R-141-3p进行功能验证mi R-141-3p在PCOS模型组卵巢中的表达水平呈显著性下调。MTT法显示,过表达mi R-141-3p后,细胞活力明显增强;干扰mi R-141-3p功能后,细胞活力明显减弱。流式细胞术显示,过表达mi R-141-3p后,细胞促凋亡的能力明显减弱;干扰mi R-141-3p功能后,细胞促凋亡的能力明显增强。生物信息学分析软件预测结果显示死亡相关蛋白激酶1(death-associated protein kinase,DAPK1)的3’-UTR区含有可与mi RNA-141-3p互补结合的序列,DAPK1可能是mi R-141-3p的靶基因。荧光定量PCR结果显示,与对照组相比,模型组大鼠卵巢中mi R-141-3p显著性下调,而DAPK1显著性上调,二者在PCOS大鼠卵巢中的表达呈明显负相关。使用mi R-141-3p模拟剂和抑制剂转染大鼠卵巢颗粒细胞后,DAPK1 m RNA和蛋白水平均与mi R-141-3p表达水平呈负相关。双荧光素酶报告基因实验显示,当HEK 293T细胞转染含Wt-DAPK1 3’-UTR的荧光素酶报告载体时,mi R-141-3p模拟剂组的荧光素酶活性较对照组明显降低;而当HEK 293T细胞转染含Mut-DAPK1 3’-UTR的荧光素酶报告载体时,mi R-141-3p模拟剂组和对照组的荧光素酶活性无显著性差异。上述结果表明,mi R-141-3p能与DAPK1基因3’-UTR结合,并对DAPK1转录有负性调控作用,证实DAPK1是mi R-141-3p的靶基因。结果表明,PCOS大鼠模型卵巢中mi RNA的异常表达可能是引起PCOS发病的重要原因。PCOS大鼠模型卵巢中低表达的mi R-141-3p通过靶向调控DAPK1的表达,进而在PCOS发生发展中发挥促进细胞凋亡的作用,mi R-141-3p/DAPK1有望成为新的PCOS诊断标志物和治疗靶点。
[Abstract]:Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrine disorder syndrome. The clinical manifestations of the patients with.PCOS, Kaohsiung, hyperinsulinemia, insulin resistance and persistent anovulatory, are diverse, and their close and long-term complications have a greater impact on the physical and mental health of women. It is reported that PCOS is the most common cause of anovulatory infertility. In addition, the risk of type 2 diabetes, cardiovascular disease and endometrial cancer in PCOS patients is significantly higher than that of non PCOS patients. In view of the great harm of PCOS to women's health, the study of its pathogenesis has become the focus of the whole gynecologic endocrinologist. The possible causes of PCOS include excessive androgen formation, hypothalamic pituitary dysfunction, insulin resistance, apoptosis and follicular development. The theory of apoptosis and follicle development caused by abnormal follicle development is concerned by more and more scholars, but the specific pathogenesis needs to be further excavated. Small RNA (micro) RNA, MI RNA) is a class of endogenous single chain, non coded small molecule RNA, with a general length of 18-24 nucleotides, which is negatively regulated by the target gene 3 'non coding region (untranslated region, UTR). In recent years, it has been found that MI RNA is expressed in female reproductive organs such as uterus, fallopian tubes, and ovaries. The extensive participation in regulating the growth and maturation of female follicles, fertilization, implantation and embryo development plays an important role in maintaining the normal reproductive function of women. At the same time, the abnormal expression of MI RNA may be related to the occurrence of many female diseases, such as the abnormal expression of MI RNA in the follicle granulocyte of PCOS patients, and one of them The abnormal expression of MI RNAs (such as let-7a, let-7i and MI R-92b) may be related to the apoptosis of follicle granulosa cells. Thus, MI RNA may affect the occurrence of PCOS by affecting the apoptosis of granulosa cells. The purpose of this study is to reveal the MI RNA and the mechanism of the regulation of the pathogenesis of PCOS. In this study, the PCOS rat model was constructed, and the MI RNA depth sequencing, fluorescence quantitative PCR, Western Blot, gene transfection and double luciferase reporter gene detection were used to excavate mi RNAs and its downstream regulation pathway related to the occurrence of PCOS disease. The main contents are as follows: 1.PCOS rats PCOS rat model.PCOS model group was established by letrozole gavage: PCOS (kg? D) was dissolved into 1% carboxymethyl cellulose (CMC) in the future for 23 days. The control group: CMC 1mg/ (kg? D) daily for 23 days. Results: after letrozole gavage, the rat vagina smear showed the control group big The estrous cycle of the rats was changed, and the estrous cycle of the model group was lost regularly. A large number of leukocytes were found in the vaginal smear of the model group, suggesting that the rats were in the estrus interval. The ovarian tissue HE staining showed that the ovarian structure was disorder, the follicle in the ovary expanded in the ovary, and the follicle in the corpus luteum and the developmental stage. The.ELISA detection showed that the concentration of serum LH, FSH, and T in the model group was significantly higher than that of the control group, and the concentration of E2 was significantly lower than that of the control group. The above experimental data indicated that we successfully constructed the PCOS rat model.2. depth sequencing and bioinformatics analysis to extract the total RNA of the ovarian tissue of the PCOS model group and the control group, and were sequenced by deep sequencing. There were 129 significant differences in the expression of MI RNAs, of which 49 were up - regulated mi RNAs, and 80 down regulated mi RNAs. The MI RNAs, which was associated with cell proliferation and apoptosis, was selected. The fluorescence quantitative PCR results showed that MI R-34b-5p. Mi R-201-5p was significantly up-regulated in the rat ovarian tissue of the model group, which further verified the accuracy of the depth sequencing results. Then, 4 differentially expressed mi RNAs were predicted by bioinformatics software, and 2060 target genes were detected. Gene Ontology (GO), Pathway analysis and other software were used for these target genes. The potential downstream regulation pathways were enriched and screened, and 4 target genes differentially expressed in MI RNAs were involved in meiosis, MAPK signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway and Notch signaling pathway, reproductive process and apoptosis,.3. selected mi R-141-3p to perform functional verification of MI R-141-3p mi R-141-3p in PCOS The expression level in the ovaries of the model group showed a significant downregulation by.MTT method. After overexpression of MI R-141-3p, the cell viability was obviously enhanced. After interfering with the function of MI R-141-3p, the cell viability was markedly weakened. The flow cytometry showed that the ability to promote apoptosis after overexpression of MI R-141-3p was obviously weakened; and the ability to promote apoptosis after MI R-141-3p function was interfered with the function of MI R-141-3p. The results of the bioinformatics analysis software predicted that the 3 '-UTR region of the death related protein kinase 1 (death-associated protein kinase, DAPK1) contains a sequence that can be complementary to MI RNA-141-3p, and DAPK1 may be the target gene of MI R-141-3p. -141-3p significantly downregulated and DAPK1 significantly increased. The expression of two in the ovary of PCOS rats was negatively correlated. DAPK1 m RNA and protein levels were negatively correlated with the MI R-141-3p expression level after transfecting rat ovarian granulosa cells with MI R-141-3p analogue and inhibitor. The double luciferase reporter gene experiment showed that HEK 293T was thin. When cell transfected with Wt-DAPK1 3 '-UTR luciferase reporter carrier, the luciferase activity of MI R-141-3p analogue group was significantly lower than that of the control group, but when HEK 293T cells transfected to the luciferase reporter carrier containing Mut-DAPK1 3' -UTR, there was no significant difference between the luciferase activity of the MI R-141-3p simulation group and the control group. Mi R-141-3p can be combined with DAPK1 gene 3 '-UTR and has a negative regulation on DAPK1 transcription. It is proved that DAPK1 is the target gene of MI R-141-3p. The results show that the abnormal expression of MI RNA in the ovaries of the PCOS rat model may be an important cause of the pathogenesis of PCOS, and the low expression of the rat model ovary is regulated by the target. The expression of PCOS R-141-3p/DAPK1 may play an important role in promoting apoptosis in the development of MI. R-141-3p/DAPK1 is expected to become a new diagnostic marker and therapeutic target for PCOS.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75;R-332

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