本文選題：miR-99a　切入點：EOC　出處：《山東大學》2015年博士論文

【摘要】：根據2014年癌癥統(tǒng)計,卵巢癌是美國女性第五位致死的病因,全年發(fā)病人數(shù)21980,但死亡人數(shù)卻在14270。由于缺乏特異性的腹盆腔癥狀和敏感標記物使得卵巢癌的早期診斷非常困難,當最終確診時絕大多數(shù)患者都已經進展為晚期。因此,卵巢癌的死亡率居高不下,預后極差,5年預期總生存率往往不到30%。為了早期診斷及改善卵巢癌的預后,非常迫切需要尋找能夠輔助早期診斷卵巢癌,并有助于選擇最佳的、個性化的治療方案的腫瘤預測標記物。MicroRNAs (miRNAs)是近年來的研究熱點,它是一組短鏈、非編碼的單鏈RNAs,直接作用于靶向mRNA的非翻譯區(qū)(Untranslated Regions, UTR)的堿基修復,成為了新的轉錄后調節(jié)器。研究表明,miRNAs參與許多生理過程,譬如細胞循環(huán)、代謝作用、血管發(fā)生、分化、細胞調亡等。并且miRNAs的異常表達可能與腫瘤有關,其通過調節(jié)基因和信號途徑參與腫瘤的發(fā)生、進展、轉移和耐藥。最為可喜的是,已有證據表明miRNA在細胞質中產生,不僅可以影響細胞的功能,也可以釋放入血液,來發(fā)揮調節(jié)遠處靶基因的功能。與其他循環(huán)中的核酸相比,循環(huán)中的miRNA水平更加穩(wěn)定、具有可重復性和持續(xù)性。循環(huán)中的miRNA在多種實體瘤中被成功評估,可作為新的無創(chuàng)性的疾病早期診斷標記物。在上皮性卵巢癌(Epithelial ovarian cancer, EOC)中,miR-15a, miR-16, miR-31, miR-200家族等miRNAs表達上調,而let-7,miRNA-34 a/b/c, miRNA-100、miR-199a/214、miR-99a等niRNAs表達下調。其中的miR-99a位于與多種疾病有關的21q21上,研究表明miR-99a在肝細胞癌、前列腺癌、頭頸部鱗狀細胞癌以及膀胱癌中均表達異常。已有研究通過miRNA微芯片技術檢測,發(fā)現(xiàn)miR-99a在正常卵巢組織和卵巢癌中表達存在差異。然而,上皮性卵巢癌中miR-99a是否在血清中異常表達及其在卵巢癌中的生物學功能仍不清楚。因此,有必要驗證miR-99a在卵巢癌,特別是上皮性卵巢癌中的作用。在本研究中,我們嘗試檢測miR-99a在上皮性卵巢癌臨床標本和細胞系中的表達,此外,通過體外研究纖維細胞生長因子受體3(Fibroblast growth factor receptor 3, FGFR3),來推測miR-99a在上皮性卵巢癌中的調控機制。第一部分：miR-99a在上皮性卵巢癌患者血清及其卵巢癌組織中的表達研究目的：檢測miR-99a在上皮性卵巢癌患者血清、卵巢癌組織及卵巢癌細胞中的表達,探討miR-99a作為上皮性卵巢癌血清標記物的可能性。研究方法：1.留取15例卵巢癌患者及3例正常志愿者血液樣本,采用實時熒光定量PCR檢測血清中miR-99a的表達。2.留取手術切除的組織標本,同樣的方法檢測miR-99a在卵巢癌組織中的表達情況。3.同法檢測卵巢癌細胞株SKOV3和正常卵巢組織細胞株OSE中miR-99a的表達。研究結果：1.卵巢癌患者的血清中miR-99a的表達水平下調。2.卵巢癌患者的卵巢癌組織中miR-99a的表達水平下調。3.卵巢癌細胞系中miR-99a表達水平下調。結論：1.miR-99a在EOC中的表達下調,提示miR-99a可能是卵巢癌的一種抑癌基因。2.在卵巢癌患者血清中miR-99a表達下調,有望成為早期診斷上皮性卵巢癌的新的生物學標記物。第二部分：miR-99a靶點基因FGFR3的預測及驗證研究目的：通過生物學信息法預測miR-99a調控的靶基因,在卵巢癌細胞中進一步驗證miR-99a如何作用于靶基因,從而為進一步闡明卵巢癌發(fā)病機制提供理論依據。研究方法：1.使用miRNA靶標預測軟件數(shù)據庫(PicTar、TargetScan、miRanda)尋找初步預測的靶基因,并將初測的基因進行RNA雜交(http://bibiserv. techfak.uni-bielefeld.de/rnahybrid/) 。2采用RT-PCR和Western Blot法檢測了OSE細胞和SKOV3細胞中FGFR3的mRNA和蛋白表達水平,從而來驗證FGFR3基因是否與EOC相關。3.構建FGFR3的3'-UTR區(qū)的熒光素酶表達質粒pGL3-FGFR3,與miR-99a共同轉染細胞,相對于無處理組(No-treated)和突變組(pGL3-mut),來觀察miR-99a對pGL3-FGFR3的熒光素酶活性的影響,進而判斷miR-99a是否可直接調控FGFR3的3'-UTR區(qū)。4.為了研究miR-99a對FGFR3的表達水平的影響,我們在SKOV3細胞中瞬時轉染miR-99a,通過qRT-PCR檢測miR-99a轉染組細胞中miR-99a的表達水平升高。然后,我們通過RT-PCR、qRT-PCR及Western Blot檢測miR-99a過表達對FGFR3的mRNA和蛋白表達水平的影響。進一步驗證兩者的關系。研究結果：1.通過生物學信息法分析預測FGFR3是miR-99a的靶基因。2.FGFR3在SKOV3細胞中相比OSE細胞的表達水平顯著升高。3.miR-99a調控FGFR3的3'-UTR區(qū)。4.miR-99a下調FGFR3的mRNA和蛋白表達水平。結論：1FGFR3是miR-99a的靶基因。2.miR-99a可下調卵巢癌細胞中FGFR3的表達。這可能是卵巢癌發(fā)生的分子生物學機制之一研究目的：探討miR-99a對卵巢癌細胞的影響；敲減FGFR3對卵巢癌細胞SKOV3增殖的影響。研究方法：1.采用CCK-8法檢測miR-99a過表達對SKOV3細胞增殖的影響。2.采用siRNA技術是將卵巢癌細胞中的FGFR3的表達降低。3.采用CCK-8法檢測轉染siRNA靶向敲減FGFR3后對SKOV3細胞增殖的影響。研究結果：1miR-99a過表達抑制卵巢癌細胞增殖。2.siRNA有效靶向敲減FGFR3表達后,卵巢癌細胞增殖被顯著抑制。結論：miR-99a通過靶向調節(jié)FGFR3抑制卵巢癌細胞的細胞增殖。
[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.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.31

【參考文獻】

相關期刊論文 前1條

1 ;MicroRNAs in neural cell development and brain diseases[J];Science China(Life Sciences);2011年12期

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本文編號：1680811