子宫肌瘤差异表达MicroRNA的筛选及miR-15b-5p调控子宫肌瘤生物学行为的机制研究

发布时间：2018-05-10 23:04

本文选题：子宫肌瘤 + 子宫肌层　；参考：《郑州大学》2016年博士论文

【摘要】：子宫平滑肌瘤(uterine leiomyomas)是育龄期女性生殖系统最常见的良性肿瘤,育龄期妇女平均发病率约为77%,而45岁女性的发病率则高达60%,且发病率呈逐年上升的趋势。子宫肌瘤典型的临床表现包括:经量过多、经期紊乱及以头晕、血色素降低为表现的继发性贫血症状;因瘤体肿大造成的盆腔压迫症状,如尿频、尿急、腹痛等;严重的子宫肌瘤会导致女性不孕和习惯性流产等生育力低下的症状,是妇科住院及全子宫切除的首要原因,浪费大量卫生资源,严重威胁妇女的身心健康。而子宫肌瘤的发病机制至今不清,是国内外研究的热点之一。微小核糖核酸(micro ribonucleic acid,miRNAs)是一种广泛存在于真核生物细胞中,大小约为21-23个碱基的非编码单链小分子核糖核酸(ribonucleic acid,RNA),在生物体内发挥重要作用,如细胞增殖,凋亡,应激反应,甚至肿瘤发病等。降解靶基因的信使核糖核酸(message RNA,m RNA)或者抑制靶基因的翻译最终沉默靶基因表达,是细胞内普遍存在的一种精细调节蛋白表达的转录后调控机制。Mi RNA参与多种肿瘤的发病过程,如细胞增殖、凋亡、炎症反应、血管生成、组织更新和抑癌致癌基因修饰,与肿瘤预后密切相关,是肿瘤的早期诊断及其预后判断的良好的生物标记物。Mi RNA参与子宫肌瘤发病的作用机制的研究尚处于初始阶段。多篇报道显示,子宫肌瘤和子宫肌层中存在差异表达显著的miRNA。检测子宫肌瘤和子宫肌层miRNA的差异表达,进而深入探讨差异miRNA对肌瘤细胞的生物学功能和调控,为揭示miRNA参与子宫肌瘤的发病机制奠定良好基础。目前,miRNA的研究方法主要有荧光实时定量PCR(Real time-PCR)、miRNA芯片及二代测序技术。而miRNA芯片是筛选子宫肌瘤和子宫肌层差异表达的miRNA的主要方法和经典手段。第一部分:子宫肌瘤组织差异表达micro RNA的筛选研究目的本研究通过miRNA芯片筛选子宫肌瘤和配对的子宫肌层样本中差异表达的miRNA,芯片联合生物信息学分析,预测差异表达显著的miRNA的靶基因及其生物学功能。方法收集2012年11月至2013年1月在郑州大学第三附属医院妇产科因子宫肌瘤行子宫全切术的10例育龄期子宫肌瘤患者的肌瘤组织和配对的肌层组织为研究对象。采用Agilent Human miRNA(8*60K)V19.0芯片筛选3例子宫肌瘤和配对的肌层组织中差异表达显著的miRNA,芯片结果用Agilent配套软件Genespring进行数据预处理和差异分析,采用聚类软件MEV进行聚类分析。应用Real time-PCR在10例子宫肌瘤和配对的肌层组织中验证hsa-miR-15b-5p、hsa-miR-21-5p、hsa-miR-34a-3p、hsa-miR-34a-5p的差异表达。利用Mi RDB、Tar Base、Target Scan、RNA22、Target Miner数据库预测上述4个miRNA的靶基因,采用基因本体论(Gene Ontology,GO)和KEGG信号通路分析(Kyoto Encyclopedia of Genes and Genomes,KEGG Pathway)探寻靶基因可能参与的生物学过程和相关信号通路。结果1、发现人子宫肌瘤和子宫肌层组织中45个差异表达的miRNA,其中19个miRNA在子宫肌瘤中表达升高,26个miRNA在子宫肌瘤中表达下降。2、验证miR-15b-5p、miR-34a-5p、miR-34a-3p、miR-21-5p在子宫肌瘤组织表达显著升高,与芯片结果基本一致,P均0.0001。3、子宫肌瘤和肌层差异表达的miRNA的靶基因主要参与形态发育、细胞生长发育等30个生物学过程。4、子宫肌瘤和肌层差异表达的miRNA的靶基因主要参与癌症、丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)、内噬作用等25个信号通路。结论子宫肌瘤和正常子宫肌层组织中存在差异表达显著的miRNA。第二部分:miR-15b-5p过表达和低表达对子宫肌瘤细胞生物学特征的影响目的根据第一部分研究结果,选择子宫肌瘤组织中表达显著升高的miR-15b-5p,通过基因转染技术,使miR-15b-5p在各型肌瘤细胞和肌层细胞中过表达和低表达,并观察miR-15b-5p低表达对子宫肌瘤细胞增殖的影响,为后续全面研究miR-15b-5p的生物学功能及远期探寻其下游靶基因提供铺垫。方法采集于2013年8月-2014年2月在美国麻省总院(Massachusetts General Hospital MGH)妇产科生殖内分泌中心因子宫肌瘤行子宫全切术的育龄期患者的肌瘤和配对的肌层组织共20例,部分消化和原代细胞分离培养为子宫肌瘤细胞(p LYO cells)和子宫肌层细胞(p MYO cells)。同时体外培养子宫肌瘤细胞系(cp LYO cells)和子宫肌层细胞系(cp MYO cells)。Real time-PCR检测不同细胞miR-15b-5p的本底表达水平。脂质体在各型细胞瞬时转染miR-15b-5p的模拟物(mimic)、抑制物(inhibitor)及其阳性、阴性对照,应用Real time-PCR验证miR-15b-5p在各组表达情况,建立miR-15b-5p高表达或低表达的模式。采用噻唑蓝实验(methylthiazolyldiphenyl-tetrazolium,MTT)检测miR-15b-5p对原代子宫肌瘤细胞增殖能力的影响。结果1、miR-15b-5p在各型细胞中本底表达情况:原代子宫肌瘤细胞中miR-15b-5p的表达水平显著高于原代子宫肌层细胞;子宫肌瘤细胞系中miR-15b-5p的表达水平显著高于子宫肌层细胞系(配对t检验,P0.001)。2、miR-15b-5p mimic转染各型细胞24和48h,各型细胞中miR-15b-5p的表达显著升高(配对t检验,P0.001)。3、miR-15b-5p inhibitor转染各型细胞24和48h,各型细胞中miR-15b-5p表达显著降低(配对t检验,P0.001)。4、原代子宫肌瘤细胞和子宫肌瘤细胞系中,不同浓度转染miR-15b-5p inhibitor组与转染其阴性对照组相比,细胞增殖率显著降低(配对t检验,P0.05)。结论miR-15b-5p可促进子宫肌瘤细胞的增殖能力。第三部分:miR-15b-5p调控子宫肌瘤细胞生物学特征的下游靶蛋白研究目的预测并验证miR-15b-5p在子宫肌瘤细胞的下游靶基因/靶蛋白,检测miR-15b-5p的下游靶蛋白在子宫肌瘤组织和子宫肌层中的表达差异,进一步揭示miR-15b-5p参与子宫肌瘤发病的分子机制。方法利用生物信息学数据库Mi RDB、Tar Base、Target Scan、RNA22、Target Miner联合预测miR-15b-5p的靶基因。转染miR-15b-5p的mimic,Real time-PCR验证预测的miR-15b-5p的靶基因RECK(Reversion-inducing cysteine-rich protein with Kazal motifs)在各型细胞的m RNA的表达水平;转染miR-15b-5p的mimic和inhibitor及各自阴性对照,免疫印迹实验(Western Blot)验证RECK蛋白在各型细胞的表达水平;转染miR15b-5p inhibitor及其阴性对照,细胞免疫荧光(Immunoflunence,IF)检测原代肌瘤及肌层细胞RECK蛋白的表达变化。继而采用Western Blot和免疫组织化学染色法(Immunohistochemical staining,IHC)检测RECK在肌瘤和肌层组织的表达差异及RECK蛋白的细胞定位。结果1、miR-15b-5p mimic转染各型细胞24小时,与空白对照组相比,肌瘤细胞系和肌层细胞系RECK m RNA表达下降(配对t检验,P0.05,P0.001)。miR-15b-5p mimic转染各型细胞48小时,与空白对照组相比,子宫肌瘤细胞系、子宫肌层细胞系及原代子宫肌瘤细胞RECK m RNA表达下降(配对t检验,P0.01,P0.001,P0.001)。2、使用10n M的miR-15b-5p mimic和inhibitor及各自阴性对照转染各型细胞48h。子宫肌瘤细胞系和原代子宫肌瘤细胞系转染miR-15b-5p mimic组与其阴性对照组相比,RECK蛋白表达显著下降;转染miR-15b-5p inhibitor组RECK蛋白表达显著升高,差异具有统计学意义(配对t检验,P均0.001)。子宫肌层细胞系转染miR-15b-5p mimic组与其阴性对照组相比,RECK蛋白表达显著下降(配对t检验,P0.001);转染inhibitor组RECK蛋白表达显著升高,差异具有统计学意(配对t检验,P0.01)。原代子宫肌层细胞转染mimic RECK蛋白表达显著下降;转染inhibitor组RECK蛋白表达显著升高,差异具有统计学意义(配对t检验,P0.05)。3、使用40n M的miR-15b-5p inhibitor和其阴性对照分别转染原代子宫肌瘤细胞和原代肌层细胞,转染inhibitor组转绿色荧光标记的RECK蛋白表达增多。4、RECK蛋白在子宫肌层组织表达显著高于配对的子宫肌瘤组织(配对t检验,P0.001)。RECK蛋白在子宫肌层细胞表达显著高于配对的子宫肌瘤细胞(配对t检验,P0.05)。5、RECK蛋白表达位于子宫肌纤维细胞的胞浆,配对的子宫肌层组织较子宫肌瘤组织中RECK蛋白表达显著升高(非配对t检验,P0.05)。结论miR-15b-5p通过调控RECK参与子宫肌瘤发病。
[Abstract]:Uterine leiomyoma (uterine leiomyomas) is the most common benign tumor of reproductive system in women of childbearing age. The average incidence of women in childbearing age is about 77%, while the incidence of 45 year old women is up to 60%, and the incidence is increasing year by year. The typical clinical manifestations of uterine myoma include: excessive menstruation, menstrual disorder and dizziness and hemoglobin. The symptoms of secondary anemia low, such as the pelvic cavity pressure caused by tumor enlargement, such as frequency of urination, urgency of urine and abdominal pain, and severe uterine fibroids can lead to low fertility symptoms such as female infertility and habitual abortion. It is the primary cause of gynecologic inpatient and total hysterectomy, wasting a large amount of health resources and seriously threatening the physical and mental health of women. The pathogenesis of uterine myoma is not clear, it is one of the hot spots at home and abroad. Micro ribonucleic acid (miRNAs) is a kind of non coding single strand small molecule ribonucleic acid (ribonucleic acid, RNA), which is widely used in eukaryotic cells and is about 21-23 bases in size, and plays an important role in the organism. Such as cell proliferation, apoptosis, stress response, and even tumor disease. Message RNA (m RNA) degrading target gene or the translation of target gene in the final silence target gene expression is a post transcriptional regulation mechanism of fine regulation protein expression,.Mi RNA involved in the pathogenesis of various tumors. Cell proliferation, apoptosis, inflammatory reaction, angiogenesis, tissue regeneration and tumor suppressor gene modification are closely related to the prognosis of tumor. It is a good biomarker for the early diagnosis and prognosis of tumor and the study of the mechanism of.Mi RNA in the pathogenesis of uterine myoma is still in the initial stage. The differential expression of differential expression of miRNA. in myoma and myometrium miRNA in myoma was found in the myometrium, and further explored the biological function and regulation of differential miRNA on myoma cells, which lay a good foundation for revealing the mechanism of miRNA to participate in the pathogenesis of uterine myoma. At present, the research methods of miRNA are mainly real-time quantitative quantitative PCR (Real Ti). Me-PCR), miRNA chip and two generation sequencing technology. MiRNA chip is the main method and classical means to screen the differential expression of uterine myoma and uterine myometrium. Part 1: the screening of differential expression of micro RNA in uterine myoma tissue, the purpose of this study was to screen the differences of uterine myoma and paired uterine myometrium samples by miRNA chip. The expression of miRNA, chip combined bioinformatics analysis, prediction of differentially expressed miRNA target genes and their biological functions. Methods from November 2012 to January 2013, the myoma and paired myoma group of 10 patients with uterine myoma at the Third Affiliated Hospital of Zhengzhou University with hysteromyoma underwent hysteromyoma The Agilent Human miRNA (8*60K) V19.0 chip was used to screen 3 cases of uterine myoma and the distinct miRNA in the paired myometrium. The chip results were pretreated with Agilent supporting software Genespring for data preprocessing and difference analysis. Cluster software MEV was used to cluster analysis. Real time-PCR was used in 10 cases of uterine myoma. The differential expression of hsa-miR-15b-5p, hsa-miR-21-5p, hsa-miR-34a-3p, and hsa-miR-34a-5p was demonstrated in the paired muscle tissue. Mi RDB, Tar Base, Target Scan, RNA22, Target databases were used to predict the above 4 target genes. Genomes, KEGG Pathway) explored the biological processes and related signaling pathways that the target gene might participate in. Results 1, 45 differentially expressed miRNA in human uterine myoma and myometrium were found, of which 19 miRNA were expressed in the uterine myoma, and 26 miRNA in the uterine fibroids decreased.2, verifying miR-15b-5p, miR-34a-5p, miR-34a-3p, miR. The expression of -21-5p in the uterine myoma tissue was significantly increased, which was basically consistent with the results of the chip, P was 0.0001.3. The target genes for the differential expression of miRNA in uterine myoma and myoma were mainly involved in 30 biological processes, such as morphological development, cell growth and development,.4. The target genes of miRNA in the differential expression of myoma and myometrium were mainly involved in cancer and mitogen activated eggs. 25 signaling pathways such as Mitogen-activated protein kinase (MAPK) and internal phagocytosis. Conclusion there is a significant difference in the expression of miRNA. second in uterine myoma and normal uterine myometrium. The effect of miR-15b-5p overexpression and low expression on the biological characteristics of uterine myoma cells is based on the results of the first part of the study and the selection of the selector The expression of miR-15b-5p in the uterine myoma tissue is significantly elevated. Through gene transfection, the expression and low expression of miR-15b-5p in various myoma cells and myometrium cells are made, and the effect of miR-15b-5p low expression on the proliferation of myoma cells is observed. The biological function of miR-15b-5p and the target gene of downstream target are explored for the future. Methods a total of 20 cases of myoma and paired myoma in the reproductive endocrine center of the gynecology and obstetrics and Gynecology of the Massachusetts General Hospital MGH, United States, in February -2014 August 2013, were collected in the reproductive endocrinology center of the gynecology and obstetrics and Gynecology of the United States, and the uterine myoma cells (P LYO cell) were isolated and cultured in partial and primary cells. S) and uterine myometrium (P MYO cells). At the same time, in vitro culture of the uterine myoma cell line (CP LYO cells) and the uterine myometrium cell line (CP MYO cells).Real time-PCR to detect the underlying expression level of different cells. In contrast, Real time-PCR was used to verify the expression of miR-15b-5p in each group and to establish a pattern of high expression or low expression of miR-15b-5p. The effect of miR-15b-5p on the proliferation of primary myoma cells was detected by methylthiazolyldiphenyl-tetrazolium (methylthiazolyldiphenyl-tetrazolium, MTT). Results 1, the expression of miR-15b-5p in various types of cells: original generation The expression level of miR-15b-5p in uterine myoma cells was significantly higher than that of the primary myometrium. The expression level of miR-15b-5p in the uterine myoma cell lines was significantly higher than that of the uterine myometrium (paired t test, P0.001).2, miR-15b-5p mimic transfected to 24 and 48h, and the expression of miR-15b-5p in each cell was significantly increased (paired t test, P0.00 1).3, miR-15b-5p inhibitor transfected cells 24 and 48h, miR-15b-5p expression in all types of cells decreased significantly (paired t test, P0.001).4, primary uterine myoma cells and uterine myoma cell lines, the proliferation rate of different concentration transfected miR-15b-5p inhibitor group was significantly lower than that of the negative control group (paired t test, P0.05). MiR-15b-5p can promote the proliferation of uterine myoma cells. Third part: the target protein of miR-15b-5p regulating the biological characteristics of uterine leiomyoma to predict and verify the downstream target gene / target protein of miR-15b-5p in the uterine myoma cells, and to detect the downstream target protein of miR-15b-5p in the uterine myoma tissue and the myometrium of the uterus. To further reveal the molecular mechanism of miR-15b-5p participation in the pathogenesis of uterine myoma. Methods using the bioinformatics database Mi RDB, Tar Base, Target Scan, RNA22, and Target Miner combined to predict the miR-15b-5p target gene. Ine-rich protein with Kazal motifs) expression level of M RNA in all types of cells; mimic and inhibitor and their negative controls for transfection of miR-15b-5p; immunoblotting experiments (Western Blot) verified the expression level of the protein in various cells; transfection and negative control, cell immunofluorescence detection The expression of RECK protein in primary myoma and myometrium cells. Then Western Blot and immunohistochemical staining (Immunohistochemical staining, IHC) were used to detect the difference of expression of RECK in myoma and myometrium and the cell location of RECK protein. Results 1, miR-15b-5p mimic was transferred to each type of cells for 24 hours, compared with the blank control group, the muscle was compared with the blank control group. The expression of RECK m RNA in the tumor cell line and the myometrium cell line decreased (paired t test, P0.05, P0.001).MiR-15b-5p mimic transfected for 48 hours. Compared with the blank control group, the uterine myoma cell line, the myometrium cell line and the original uterine myoma cells decreased the RECK m RNA expression. The expression of RECK protein in the 48h. uterine myoma cell lines and the primary uterine myoma cells transfected by b-5p mimic and inhibitor and their negative control cells transfected to miR-15b-5p mimic group was significantly lower than that of the negative control group. The expression of RECK protein in the miR-15b-5p inhibitor group was significantly higher than that of the miR-15b-5p inhibitor group (paired t test, P). The expression of RECK protein in the miR-15b-5p mimic group was significantly decreased (paired t test, P0.001), and the expression of RECK protein in the transfected inhibitor group was significantly higher than that of the negative control group. The difference was statistically significant (paired t test, P0.01). The expression of mimic RECK protein transfected in the primary myometrium cells decreased significantly; the expression of mimic RECK protein was significantly decreased; the transfection of the primary myometrium cells transfected to the inhibitor group was significantly decreased. The expression of RECK protein in group inhibitor was significantly higher, and the difference was statistically significant (paired t test, P0.05).3. 40n M miR-15b-5p inhibitor and its negative control were used to transfect the primary myoma cells and primary myometrium cells respectively. The RECK protein expressed in the inhibitor group was increased to the RECK protein, and the protein was in the myometrium of the uterus. The expression of the fabric was significantly higher than that of the paired uterine myoma (paired t test, P0.001), the expression of.RECK protein in the uterine myoma cells was significantly higher than that of the paired uterine myoma cells (paired t test, P0.05).5, the RECK protein expression was located in the cytoplasm of the fibroblast cells of the uterus, and the paired uterine myometrium was significantly more expressed than the RECK protein in the uterine myoma tissue. Elevated (unpaired t test, P0.05). Conclusion miR-15b-5p participates in the pathogenesis of uterine fibroids by regulating RECK.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

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