X染色体基因调控的microRNA在特纳综合征中作用和机制研究

发布时间：2018-04-30 20:54

本文选题：特征综合性 + 血浆　；参考：《浙江大学》2016年博士论文

【摘要】：第一部分:45,XO特纳综合征患者血浆小RNA组学研究目的:筛选45,XO特纳综合征(TS)与正常46,XX女性差异表达的血浆microRNA(miRNA)材料与方法:收集10例未进行激素替代治疗的纯合45,X0病人血浆及10例月经周期正常的46,XX女性血浆。采用HiSeq 2000测序平台,对血浆中的小RNA进行测序。用Ingenuity Pathway Analysis软件进行生物信息学分析。对其中几种差异显著的高表达血浆miRNA,进行扩大样本量的real time quantitative PCR(qRT-PCR)验证。结果:共筛选到182种差异表达的miRNA,其中85种在45,XO组和46,XX组存在显著差异。生物信息学分析显示差异表达的miRNA,与特纳患者先天发育异常、生殖系统疾病、智力低下等异常临床表现密切相关。qRT-PCR结果进一步证实miR-320、miR-486-5p、let-7a-5p等miRNA在两组间存在显著差异。结论:小RNA组学分析显示45,XO患者较46,XX,血浆miRNA表达谱明显改变。这些改变的miRNA与45,XO异常临床表型密切相关,提示miRNA可能在其中有潜在作用。第二部分:特纳综合征差异表达的microRNA与X染色体逃逸失活基因关系研究目的:探索特纳综合征差异表达的microRNA与X染色体逃逸失活基因关系。材料与方法:检测核型为45,XO、46,XX、46,XY、47,XXY患者外周血单个核细胞(PMBC)miR-320a,miR-486-5p及逃逸失活基因 KDM5C,KDM6A,DDX3X的表达水平,探索其潜在的关联性。同时在核型为45,XO、46,XX流产胚胎性腺组织中验证。通过体外细胞实验,验证前面发现的microRNA与X染色体逃逸失活基因关系。结果:与血浆结果一致,miR-320a,miR-486-5p在45,XO的PMBC和流产胚胎性腺组织中高表达。通过45,XO、46,XX、46,XY、47,XXY四组比较,发现miR-320a,miR-486-5p与性染色体数目成负相关。同时在四组中验证了具有X/Y同源基因的KDM5C为逃逸失活基因,其在45,XO的PMBC和流产胚胎性腺组织中低表达,与miR-320a,miR-486-5p表达存在负相关。体外细胞实验,发现干扰KDM5C 后,miR-320a,miR-486-5p,miR-320a 的初始产物 pri-miR-320a 高表达,而miR-486-5p的初始产物pri-miR-486-5p没有改变,提示KDM5C能够负向调控miR-320a起始转录。而miR-320a过表达后KDM5C没有改变,提示miR-320a不能调控KDM5C表达。荧光素酶报告基因及ChIP实验,证实敲低作为转录抑制因子的H3K4me3去甲化酶KDM5C,可以解除其对编码miR-320a基因的启动子特定区段抑制作用,使该区域转录激活标志H3K4me3水平升高,miR-320a表达增加。结论:PMBC和流产胚胎性腺组织中,45,XO的miR-320a,miR-486-5p高表达,且与X染色体逃逸失活基因KDM5C水平呈负相关。敲低KDM5C后,能够通过增加miR-320a启动子特定区域H3K4me3的水平,反式激活miR-320a的转录。第三部分:X染色体基因调控的microRNA在特纳综合征先天性腺发育不全中作用研究目的:探索miR-320a及KDM5C在特纳综合征先天性腺发育不全中的作用。材料与方法:在原代培养的胎儿卵巢细胞(HFO)中,分别转染miR-320a的模拟物及KDM5C小干扰RNA,检测与卵巢发育相关因子的改变。同时通过荧光素酶报告基因验证软件预测的miR-320a靶点。结果:HFO细胞,分别转入miR-320a的模拟物及KDM5C小干扰RNA后,KITLG和BMP5的表达水平明显下降。转染miR-320a的抑制物后,KDM5C小干扰RNA对KITLG的抑制作用明显减弱。软件和荧光素酶报告基因证实KITLG是miR-320a的直接作用靶点。westerone blot实验发现45,XO的流产胎儿性腺组织的KITLG水平明显低于46,XX。结论:卵巢发育重要因子KITLG是miR-320a的直接作用靶点,上调的miR-320a及干扰KDM5C均可使其表达下调。其可能在特纳先天性性腺发育不全中发挥重要作用。
[Abstract]:The first part: 45, the purpose of the small RNA group study of XO Turner syndrome patients: to screen the plasma microRNA (miRNA) materials and methods of 45, XO Turner syndrome (TS) and normal 46, XX women: collect 10 cases of homozygous 45 without hormone replacement therapy, X0 patient's blood plasma and 10 cases of normal menstrual cycle 46, XX female plasma. HiSeq 2000 Sequencing and sequencing of small RNA in plasma. Bioinformatics analysis was performed with Ingenuity Pathway Analysis software. A few significant high expression of plasma miRNA and real time quantitative PCR (qRT-PCR) were tested. Results: 182 kinds of differential expression miRNA were screened, of which 85 were 45, XO and 4 6, there were significant differences in the XX group. Bioinformatics analysis showed that differential expression of miRNA was closely related to abnormal clinical manifestations of congenital dysplasia, reproductive system disease and mental retardation in Turner patients..qRT-PCR results further confirmed that miR-320, miR-486-5p, let-7a-5p and miRNA were significantly different among the two groups. Conclusion: small RNA analysis showed that there was a significant difference between groups. 45, XO patients are more than 46, XX, and plasma miRNA expression profiles. These changes of miRNA are closely related to abnormal clinical phenotypes of 45 and XO, suggesting that miRNA may have potential roles. The second part: Study on the relationship between microRNA and X chromosomal escape inactivation genes differentially expressed by Turner syndrome: to explore the differentially expressed microRNA syndrome of Turner syndrome X chromosome escape inactivation genes. Materials and methods: to detect the expression level of KDM5C, miR-486-5p and escape inactivation genes KDM5C, KDM6A, DDX3X in peripheral blood mononuclear cells (PMBC) of patients with 45, XO, 46, XX, 46, XY, 47, XXY, and explore the potential correlation in the karyotype 45, XO, 46, abortion embryo gonadal tissue. In vitro cell test, the relationship between microRNA and X chromosome escape inactivation genes was verified. Results: in accordance with the plasma results, miR-320a, miR-486-5p were highly expressed in 45, XO PMBC and abortion embryo gonadal tissues. Through 45, XO, 46, XX, XY, 47, XXY four found miR-320a, miR-486-5p and sex chromosomes were negatively correlated. In four groups, the X/Y homologous KDM5C was proved to be an escape inactivation gene, which was low expression in 45, XO PMBC and aborted embryo gonadal tissue, and negative correlation with miR-320a, miR-486-5p expression. In vitro cell experiment, after the disturbance of KDM5C, the initial high expression of miR-320a, miR-486-5p, miR-320a was found, and miR-486-5p The initial product pri-miR-486-5p does not change, suggesting that KDM5C can negatively regulate the transcription of miR-320a. While miR-320a overexpression, KDM5C does not change, suggesting that miR-320a does not regulate KDM5C expression. The luciferase reporter gene and ChIP experiments have proved that the H3K4me3 normethylation KDM5C, which is a transcriptional inhibitor, can be relieved of its mi coding. The inhibition action of the specific promoter of the R-320a gene makes the transcriptional activation marker H3K4me3 level and miR-320a expression increase in the region. Conclusion: in PMBC and abortion embryo gonadal tissues, 45, XO miR-320a, miR-486-5p are highly expressed and negatively correlated with KDM5C water level of the X chromosome escape inactivation gene. After knocking down KDM5C, the miR-320a can be increased by miR-320a. The level of H3K4me3 in the specific promoter region and trans activation of the transcription of miR-320a. Third part: the role of microRNA regulated by X chromosome in innate gonadoplasia of Turner syndrome: To explore the role of miR-320a and KDM5C in the congenital gonadal dysplasia of Turner syndrome. Materials and methods: in the primary culture of fetal eggs In nesting cells (HFO), miR-320a mimics and KDM5C small interference RNA were respectively transfected to detect changes in ovarian development related factors. At the same time, the miR-320a target of the software predicted by the luciferase reporter gene was verified. Results: HFO cells were transferred to miR-320a mimics and KDM5C small interference RNA, KITLG and BMP5 decreased significantly. After transfection of miR-320a inhibitor, the inhibitory effect of KDM5C small interference RNA on KITLG was obviously weakened. The software and luciferase reporter gene confirmed that KITLG was the direct target of miR-320a, the.Westerone blot experiment found 45, the KITLG level of the aborted fetal gonadal tissues of XO was obviously lower than 46, and the XX. conclusion: the important factor of ovarian development is KITLG. The direct target, the up regulation of miR-320a and the interference of KDM5C can reduce their expression, which may play an important role in Turner's congenital gonadal hypoplasia.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.1

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