miR-99a靶向调控FGFR3抑制上皮性卵巢癌细胞增殖的机制研究

发布时间：2018-03-29 10:50

本文选题：miR-99a　切入点：EOC　出处：《山东大学》2015年博士论文

【摘要】：根据2014年癌症统计,卵巢癌是美国女性第五位致死的病因,全年发病人数21980,但死亡人数却在14270。由于缺乏特异性的腹盆腔症状和敏感标记物使得卵巢癌的早期诊断非常困难,当最终确诊时绝大多数患者都已经进展为晚期。因此,卵巢癌的死亡率居高不下,预后极差,5年预期总生存率往往不到30%。为了早期诊断及改善卵巢癌的预后,非常迫切需要寻找能够辅助早期诊断卵巢癌,并有助于选择最佳的、个性化的治疗方案的肿瘤预测标记物。MicroRNAs (miRNAs)是近年来的研究热点,它是一组短链、非编码的单链RNAs,直接作用于靶向mRNA的非翻译区(Untranslated Regions, UTR)的碱基修复,成为了新的转录后调节器。研究表明,miRNAs参与许多生理过程,譬如细胞循环、代谢作用、血管发生、分化、细胞调亡等。并且miRNAs的异常表达可能与肿瘤有关,其通过调节基因和信号途径参与肿瘤的发生、进展、转移和耐药。最为可喜的是,已有证据表明miRNA在细胞质中产生,不仅可以影响细胞的功能,也可以释放入血液,来发挥调节远处靶基因的功能。与其他循环中的核酸相比,循环中的miRNA水平更加稳定、具有可重复性和持续性。循环中的miRNA在多种实体瘤中被成功评估,可作为新的无创性的疾病早期诊断标记物。在上皮性卵巢癌(Epithelial ovarian cancer, EOC)中,miR-15a, miR-16, miR-31, miR-200家族等miRNAs表达上调,而let-7,miRNA-34 a/b/c, miRNA-100、miR-199a/214、miR-99a等niRNAs表达下调。其中的miR-99a位于与多种疾病有关的21q21上,研究表明miR-99a在肝细胞癌、前列腺癌、头颈部鳞状细胞癌以及膀胱癌中均表达异常。已有研究通过miRNA微芯片技术检测,发现miR-99a在正常卵巢组织和卵巢癌中表达存在差异。然而,上皮性卵巢癌中miR-99a是否在血清中异常表达及其在卵巢癌中的生物学功能仍不清楚。因此,有必要验证miR-99a在卵巢癌,特别是上皮性卵巢癌中的作用。在本研究中,我们尝试检测miR-99a在上皮性卵巢癌临床标本和细胞系中的表达,此外,通过体外研究纤维细胞生长因子受体3(Fibroblast growth factor receptor 3, FGFR3),来推测miR-99a在上皮性卵巢癌中的调控机制。第一部分：miR-99a在上皮性卵巢癌患者血清及其卵巢癌组织中的表达研究目的：检测miR-99a在上皮性卵巢癌患者血清、卵巢癌组织及卵巢癌细胞中的表达,探讨miR-99a作为上皮性卵巢癌血清标记物的可能性。研究方法：1.留取15例卵巢癌患者及3例正常志愿者血液样本,采用实时荧光定量PCR检测血清中miR-99a的表达。2.留取手术切除的组织标本,同样的方法检测miR-99a在卵巢癌组织中的表达情况。3.同法检测卵巢癌细胞株SKOV3和正常卵巢组织细胞株OSE中miR-99a的表达。研究结果：1.卵巢癌患者的血清中miR-99a的表达水平下调。2.卵巢癌患者的卵巢癌组织中miR-99a的表达水平下调。3.卵巢癌细胞系中miR-99a表达水平下调。结论：1.miR-99a在EOC中的表达下调,提示miR-99a可能是卵巢癌的一种抑癌基因。2.在卵巢癌患者血清中miR-99a表达下调,有望成为早期诊断上皮性卵巢癌的新的生物学标记物。第二部分：miR-99a靶点基因FGFR3的预测及验证研究目的：通过生物学信息法预测miR-99a调控的靶基因,在卵巢癌细胞中进一步验证miR-99a如何作用于靶基因,从而为进一步阐明卵巢癌发病机制提供理论依据。研究方法：1.使用miRNA靶标预测软件数据库(PicTar、TargetScan、miRanda)寻找初步预测的靶基因,并将初测的基因进行RNA杂交(http://bibiserv. techfak.uni-bielefeld.de/rnahybrid/) 。2采用RT-PCR和Western Blot法检测了OSE细胞和SKOV3细胞中FGFR3的mRNA和蛋白表达水平,从而来验证FGFR3基因是否与EOC相关。3.构建FGFR3的3'-UTR区的荧光素酶表达质粒pGL3-FGFR3,与miR-99a共同转染细胞,相对于无处理组(No-treated)和突变组(pGL3-mut),来观察miR-99a对pGL3-FGFR3的荧光素酶活性的影响,进而判断miR-99a是否可直接调控FGFR3的3'-UTR区。4.为了研究miR-99a对FGFR3的表达水平的影响,我们在SKOV3细胞中瞬时转染miR-99a,通过qRT-PCR检测miR-99a转染组细胞中miR-99a的表达水平升高。然后,我们通过RT-PCR、qRT-PCR及Western Blot检测miR-99a过表达对FGFR3的mRNA和蛋白表达水平的影响。进一步验证两者的关系。研究结果：1.通过生物学信息法分析预测FGFR3是miR-99a的靶基因。2.FGFR3在SKOV3细胞中相比OSE细胞的表达水平显著升高。3.miR-99a调控FGFR3的3'-UTR区。4.miR-99a下调FGFR3的mRNA和蛋白表达水平。结论：1FGFR3是miR-99a的靶基因。2.miR-99a可下调卵巢癌细胞中FGFR3的表达。这可能是卵巢癌发生的分子生物学机制之一研究目的：探讨miR-99a对卵巢癌细胞的影响；敲减FGFR3对卵巢癌细胞SKOV3增殖的影响。研究方法：1.采用CCK-8法检测miR-99a过表达对SKOV3细胞增殖的影响。2.采用siRNA技术是将卵巢癌细胞中的FGFR3的表达降低。3.采用CCK-8法检测转染siRNA靶向敲减FGFR3后对SKOV3细胞增殖的影响。研究结果：1miR-99a过表达抑制卵巢癌细胞增殖。2.siRNA有效靶向敲减FGFR3表达后,卵巢癌细胞增殖被显著抑制。结论：miR-99a通过靶向调节FGFR3抑制卵巢癌细胞的细胞增殖。
[Abstract]:According to the 2014 cancer statistics, ovarian cancer is the fifth cause of death among women in the United States, the number of annual incidence of 21980, but the death toll is 14270. due to the lack of specific and sensitive markers of pelvic and abdominal symptoms makes early diagnosis of ovarian cancer is very difficult, when the final diagnosis most patients have progressed to advanced. Therefore, high the mortality rate of ovarian cancer, prognosis, survival rate of 5 years is expected to total more than 30%. in order to improve the early diagnosis and prognosis of ovarian cancer, so there is an urgent need to find can assist in early diagnosis of ovarian cancer, and help to choose the best treatment plan, personalized tumor prediction marker.MicroRNAs (miRNAs) is a research hotspot in recent years here, it is a group of short chain, single stranded RNAs encoding, direct role in targeting the untranslated region of mRNA (Untranslated Regions UTR) alkali motonaga complex, has become a new turn The regulator after the record. The results show that miRNAs is involved in many physiological processes, such as cell cycle, metabolism, angiogenesis, cell differentiation, apoptosis and abnormal expression. MiRNAs may be associated with the tumor, its involvement in tumor by regulating genes and signaling pathways in the occurrence, development, metastasis and drug resistance. The most gratifying is that there is evidence that miRNA is produced in the cytoplasm, not only can affect cell function, can be released into the blood, to regulate the distance of target gene function. Compared with other nucleic acid cycle, circulating levels of miRNA in the more stable, repeatable and persistent in the circulation of miRNA was successfully. In the evaluation of a variety of solid tumors, can be used as a new noninvasive biomarkers for early diagnosis of disease. In epithelial ovarian cancer (Epithelial ovarian, cancer, EOC), miR-15a, miR-16, miR-31, miRNAs and other miR-200 family expression, Let-7, miRNA-34 a/b/c, miRNA-100, miR-199a/214, miR-99a, niRNAs expression. The miR-99a in 21q21 is associated with many diseases, studies have shown that miR-99a in hepatocellular carcinoma, prostate cancer, head and neck squamous cell carcinoma and bladder carcinoma were abnormal expression. Have research on micro chip technology to detect by miRNA, found miR-99a there are differences in the expression of normal ovarian tissue and ovarian cancer. However, epithelial ovarian cancer miR-99a abnormal expression and its biological function in ovarian cancer in serum is still unclear. Therefore, it is necessary to verify miR-99a in ovarian cancer, especially in epithelial ovarian cancer. In this study, we try to detect expression of miR-99a in epithelial ovarian cancer cell lines and clinical specimens in addition, through the study of fiber cell growth factor receptor 3 (Fibroblast growth factor receptor 3, FGFR3) To speculate, the regulatory mechanism of miR-99a in epithelial ovarian carcinoma. The first part: To study the expression of miR-99a in epithelial ovarian carcinoma and ovarian carcinoma: detection of serum miR-99a in patients with epithelial ovarian cancer, ovarian cancer and ovarian cancer cells express, explore the possibility of miR-99a as epithelial ovarian cancer serum markers. Methods: 1. specimens from 15 patients with ovarian cancer and 3 cases of normal volunteers using blood samples, the expression of.2. miR-99a real-time fluorescence quantitative PCR detection in serum specimens from surgical resection specimens, the same method for detection of miR-99a in ovarian cancer tissue. The expression of miR-99a.3. with the detection of ovarian cancer cell line SKOV3 and normal ovarian tissue cell OSE expression. Results: the expression level of miR-99a in serum of 1. patients with ovarian cancer in patients with ovarian cancer by.2. in ovarian cancer The expression of miR-99a in tissues of the lower levels of expression of miR-99a.3. in ovarian cancer cell lines. Conclusion: the expression of 1.miR-99a in EOC, suggesting that miR-99a may be a tumor suppressor gene.2. in ovarian cancer patient serum miR-99a expression of ovarian cancer, is expected to become a new biomarker for early diagnosis of epithelial ovarian cancer the second part: Objective To study the prediction and verification of miR-99a target gene FGFR3: target gene by biological information method to predict miR-99a regulation, further validation of miR-99a how to effect on the target gene in ovarian cancer cells, from which provide a theoretical basis for further elucidating the mechanism of ovarian cancer. Methods: 1. using miRNA target prediction software the database (PicTar, TargetScan, miRanda) of target genes for preliminary prediction, RNA hybridization and initial test gene (http://bibiserv. techfak.uni-bielefel D.de/rnahybrid/.2 and Western RT-PCR) using Blot method to detect the mRNA and protein of FGFR3 OSE cells and SKOV3 cells in the expression level, to verify whether the FGFR3 gene associated with EOC.3. to construct the FGFR3 3'-UTR region of the luciferase expression plasmid pGL3-FGFR3 and miR-99a co transfected cells, compared with non treatment group (No-treated) and mutation group (pGL3-mut), to observe the effect of miR-99a on luciferase activity of pGL3-FGFR3, and then determine whether miR-99a affects 3'-UTR.4. FGFR3 region directly regulate the expression level of miR-99a in order to study FGFR3, we transfected miR-99a in SKOV3 cells, the expression level of miR-99a qRT-PCR in the detection of miR-99a transfected cells increased. Then, we RT-PCR, qRT-PCR Western and Blot miR-99a to detect the expression of mRNA and protein of FGFR3 expression level. Further analysis of the relationship between the two. Research results: 1. by the biological information method analysis and prediction of FGFR3 is mRNA and protein expression of target gene.2.FGFR3 miR-99a in SKOV3 cells compared to the expression level of OSE cells increased significantly in the regulation of FGFR3 3'-UTR.4.miR-99a.3.miR-99a down FGFR3. Conclusion: 1FGFR3 is the target gene expression of.2.miR-99a miR-99a downregulated FGFR3 in ovarian cancer cells. This may be the molecular mechanisms of ovarian cancer is one of the research objective: To investigate the effect of miR-99a on ovarian cancer cells; knockdown effect of FGFR3 on the proliferation of ovarian cancer cell line SKOV3. Methods: 1. CCK-8 method was used to detect the effect of overexpression of miR-99a on proliferation of SKOV3 cells by using.2. siRNA technology is to reduce the expression of ovarian cancer cells the effect of FGFR3 on proliferation of SKOV3 cells was detected by CCK-8.3. transfection of siRNA targeted knockdown of FGFR3. Results: the overexpression of 1miR-99a suppression Ovarian cancer cells proliferation,.2.siRNA, and effective FGFR3 knockdown of miR-99a expression significantly inhibited the proliferation of ovarian cancer cells. Conclusion: miR-99a inhibits the proliferation of ovarian cancer cells through targeted regulation of FGFR3.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.31

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