發(fā)布時(shí)間：2018-03-29 10:50

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

【摘要】：根據(jù)2014年癌癥統(tǒng)計(jì),卵巢癌是美國(guó)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(biāo)記物使得卵巢癌的早期診斷非常困難,當(dāng)最終確診時(shí)絕大多數(shù)患者都已經(jīng)進(jìn)展為晚期。因此,卵巢癌的死亡率居高不下,預(yù)后極差,5年預(yù)期總生存率往往不到30%。為了早期診斷及改善卵巢癌的預(yù)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(gè)性化的治療方案的腫瘤預(yù)測(cè)標(biāo)記物。MicroRNAs (miRNAs)是近年來(lái)的研究熱點(diǎn),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(fù),成為了新的轉(zhuǎn)錄后調(diào)節(jié)器。研究表明,miRNAs參與許多生理過(guò)程,譬如細(xì)胞循環(huán)、代謝作用、血管發(fā)生、分化、細(xì)胞調(diào)亡等。并且miRNAs的異常表達(dá)可能與腫瘤有關(guān),其通過(guò)調(diào)節(jié)基因和信號(hào)途徑參與腫瘤的發(fā)生、進(jìn)展、轉(zhuǎn)移和耐藥。最為可喜的是,已有證據(jù)表明miRNA在細(xì)胞質(zhì)中產(chǎn)生,不僅可以影響細(xì)胞的功能,也可以釋放入血液,來(lái)發(fā)揮調(diào)節(jié)遠(yuǎn)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(fù)性和持續(xù)性。循環(huán)中的miRNA在多種實(shí)體瘤中被成功評(píng)估,可作為新的無(wú)創(chuàng)性的疾病早期診斷標(biāo)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(dá)上調(diào),而let-7,miRNA-34 a/b/c, miRNA-100、miR-199a/214、miR-99a等niRNAs表達(dá)下調(diào)。其中的miR-99a位于與多種疾病有關(guān)的21q21上,研究表明miR-99a在肝細(xì)胞癌、前列腺癌、頭頸部鱗狀細(xì)胞癌以及膀胱癌中均表達(dá)異常。已有研究通過(guò)miRNA微芯片技術(shù)檢測(cè),發(fā)現(xiàn)miR-99a在正常卵巢組織和卵巢癌中表達(dá)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(dá)及其在卵巢癌中的生物學(xué)功能仍不清楚。因此,有必要驗(yàn)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(cè)miR-99a在上皮性卵巢癌臨床標(biāo)本和細(xì)胞系中的表達(dá),此外,通過(guò)體外研究纖維細(xì)胞生長(zhǎng)因子受體3(Fibroblast growth factor receptor 3, FGFR3),來(lái)推測(cè)miR-99a在上皮性卵巢癌中的調(diào)控機(jī)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(dá)研究目的：檢測(cè)miR-99a在上皮性卵巢癌患者血清、卵巢癌組織及卵巢癌細(xì)胞中的表達(dá),探討miR-99a作為上皮性卵巢癌血清標(biāo)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(shí)時(shí)熒光定量PCR檢測(cè)血清中miR-99a的表達(dá)。2.留取手術(shù)切除的組織標(biāo)本,同樣的方法檢測(cè)miR-99a在卵巢癌組織中的表達(dá)情況。3.同法檢測(cè)卵巢癌細(xì)胞株SKOV3和正常卵巢組織細(xì)胞株OSE中miR-99a的表達(dá)。研究結(jié)果：1.卵巢癌患者的血清中miR-99a的表達(dá)水平下調(diào)。2.卵巢癌患者的卵巢癌組織中miR-99a的表達(dá)水平下調(diào)。3.卵巢癌細(xì)胞系中miR-99a表達(dá)水平下調(diào)。結(jié)論：1.miR-99a在EOC中的表達(dá)下調(diào),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(dá)下調(diào),有望成為早期診斷上皮性卵巢癌的新的生物學(xué)標(biāo)記物。第二部分：miR-99a靶點(diǎn)基因FGFR3的預(yù)測(cè)及驗(yàn)證研究目的：通過(guò)生物學(xué)信息法預(yù)測(cè)miR-99a調(diào)控的靶基因,在卵巢癌細(xì)胞中進(jìn)一步驗(yàn)證miR-99a如何作用于靶基因,從而為進(jìn)一步闡明卵巢癌發(fā)病機(jī)制提供理論依據(jù)。研究方法：1.使用miRNA靶標(biāo)預(yù)測(cè)軟件數(shù)據(jù)庫(kù)(PicTar、TargetScan、miRanda)尋找初步預(yù)測(cè)的靶基因,并將初測(cè)的基因進(jìn)行RNA雜交(http://bibiserv. techfak.uni-bielefeld.de/rnahybrid/) 。2采用RT-PCR和Western Blot法檢測(cè)了OSE細(xì)胞和SKOV3細(xì)胞中FGFR3的mRNA和蛋白表達(dá)水平,從而來(lái)驗(yàn)證FGFR3基因是否與EOC相關(guān)。3.構(gòu)建FGFR3的3'-UTR區(qū)的熒光素酶表達(dá)質(zhì)粒pGL3-FGFR3,與miR-99a共同轉(zhuǎn)染細(xì)胞,相對(duì)于無(wú)處理組(No-treated)和突變組(pGL3-mut),來(lái)觀察miR-99a對(duì)pGL3-FGFR3的熒光素酶活性的影響,進(jìn)而判斷miR-99a是否可直接調(diào)控FGFR3的3'-UTR區(qū)。4.為了研究miR-99a對(duì)FGFR3的表達(dá)水平的影響,我們?cè)赟KOV3細(xì)胞中瞬時(shí)轉(zhuǎn)染miR-99a,通過(guò)qRT-PCR檢測(cè)miR-99a轉(zhuǎn)染組細(xì)胞中miR-99a的表達(dá)水平升高。然后,我們通過(guò)RT-PCR、qRT-PCR及Western Blot檢測(cè)miR-99a過(guò)表達(dá)對(duì)FGFR3的mRNA和蛋白表達(dá)水平的影響。進(jìn)一步驗(yàn)證兩者的關(guān)系。研究結(jié)果：1.通過(guò)生物學(xué)信息法分析預(yù)測(cè)FGFR3是miR-99a的靶基因。2.FGFR3在SKOV3細(xì)胞中相比OSE細(xì)胞的表達(dá)水平顯著升高。3.miR-99a調(diào)控FGFR3的3'-UTR區(qū)。4.miR-99a下調(diào)FGFR3的mRNA和蛋白表達(dá)水平。結(jié)論：1FGFR3是miR-99a的靶基因。2.miR-99a可下調(diào)卵巢癌細(xì)胞中FGFR3的表達(dá)。這可能是卵巢癌發(fā)生的分子生物學(xué)機(jī)制之一研究目的：探討miR-99a對(duì)卵巢癌細(xì)胞的影響；敲減FGFR3對(duì)卵巢癌細(xì)胞SKOV3增殖的影響。研究方法：1.采用CCK-8法檢測(cè)miR-99a過(guò)表達(dá)對(duì)SKOV3細(xì)胞增殖的影響。2.采用siRNA技術(shù)是將卵巢癌細(xì)胞中的FGFR3的表達(dá)降低。3.采用CCK-8法檢測(cè)轉(zhuǎn)染siRNA靶向敲減FGFR3后對(duì)SKOV3細(xì)胞增殖的影響。研究結(jié)果：1miR-99a過(guò)表達(dá)抑制卵巢癌細(xì)胞增殖。2.siRNA有效靶向敲減FGFR3表達(dá)后,卵巢癌細(xì)胞增殖被顯著抑制。結(jié)論：miR-99a通過(guò)靶向調(diào)節(jié)FGFR3抑制卵巢癌細(xì)胞的細(xì)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號(hào)：1680811