本文選題：Ⅰ型子宮內(nèi)膜腺癌 + miRNA-mRNA網(wǎng)絡(luò)　； 參考：《南方醫(yī)科大學(xué)》2016年博士論文

【摘要】：研究背景：子宮內(nèi)膜癌(endometrial carcinoma, EC)是子宮內(nèi)膜上皮細(xì)胞來(lái)源的一種常見(jiàn)的女性生殖系統(tǒng)惡性腫瘤,是婦科三大惡性腫瘤之一,發(fā)病率僅次于宮頸癌,位于第二位,占婦科腫瘤癌癥的30%左右。雖然相對(duì)于其它女性生殖系統(tǒng)惡性腫瘤患者,子宮內(nèi)膜癌患者大多數(shù)五年的生存率不低,預(yù)后程度也較好,但在我國(guó)有逐年上升趨勢(shì)。嚴(yán)重危害女性健康。作為一種病因尚不明確的疾病,子宮內(nèi)膜癌中有85%-90%為Ⅰ型子宮內(nèi)膜癌；目前腫瘤疾病相關(guān)的研究依舊不能很好的解釋子宮內(nèi)膜癌的發(fā)病過(guò)程。子宮內(nèi)膜癌的高發(fā)人群主要是絕經(jīng)后婦女,年齡多為50-60歲,約占總發(fā)病人數(shù)的75%左右。近年來(lái),在國(guó)內(nèi)外的相關(guān)研究報(bào)道中,子宮內(nèi)膜癌的發(fā)病率和死亡率在全世界范圍內(nèi)呈明顯年輕化和迅速上升的趨勢(shì)。美國(guó)癌癥協(xié)會(huì)的評(píng)估報(bào)告顯示,2005年美國(guó)新發(fā)現(xiàn)的子宮內(nèi)膜癌40880例,其中7310例患者死亡,2010年,有新增病例43470例,死亡7950例,而最新的流行病學(xué)調(diào)查資料則顯示,在2015年美國(guó)女性所有新發(fā)的癌癥中,子宮內(nèi)膜癌發(fā)病率明顯上升,僅排在乳腺癌、肺癌和結(jié)腸癌之后位居第4位,并且預(yù)計(jì)因子宮內(nèi)膜癌死亡的人數(shù)將達(dá)到10170之多。在我國(guó),目前還沒(méi)有大規(guī)模完全的子宮內(nèi)膜癌統(tǒng)計(jì)學(xué)資料報(bào)道,但據(jù)不完全的調(diào)查統(tǒng)計(jì)結(jié)果顯示,我國(guó)子宮內(nèi)膜癌的發(fā)病率逐年上升的趨勢(shì)已非常明顯。根據(jù)腫瘤病理形態(tài)特征不同,子宮內(nèi)膜癌分為兩種類(lèi)型：即Ⅰ型雌激素依賴(lài)和Ⅱ型非雌激素依賴(lài),兩者的發(fā)生發(fā)展的分子機(jī)制、形態(tài)學(xué)特征以及預(yù)后等存在明顯的差別。Ⅰ型與雌激素過(guò)度刺激相關(guān),主要為子宮內(nèi)膜過(guò)度生長(zhǎng)的基礎(chǔ)上發(fā)展形成的子宮內(nèi)膜樣腺癌,占子宮內(nèi)膜癌的80-90%；Ⅱ型病理類(lèi)型主要為透明細(xì)胞癌,漿液性乳頭狀癌,粘液腺癌以及腺鱗癌等,臨床以雌激素依賴(lài)的Ⅰ型為主[9],因此本研究主要針對(duì)Ⅰ型的子宮內(nèi)膜樣腺癌進(jìn)行研究,后面提到的子宮內(nèi)膜癌均指代Ⅰ型子宮內(nèi)膜樣腺癌。子宮內(nèi)膜癌臨床早期表現(xiàn)為陰道不規(guī)則流血癥狀,可有助于早期診斷,并且80%子宮內(nèi)膜癌患者的病灶會(huì)局限在子宮體內(nèi),因此,相對(duì)于其它的女性生殖系統(tǒng)惡性腫瘤患者,子宮內(nèi)膜癌患者大多數(shù)五年的生存率不低,預(yù)后程度也較好,有研究報(bào)道,早期子宮內(nèi)膜癌患者的5年無(wú)病存活率可達(dá)98%,5年總生存率可達(dá)93%。然而,仍然有部分子宮內(nèi)膜癌患者的病理分化程度低,侵襲轉(zhuǎn)移率高,預(yù)后效果差,通常會(huì)因?yàn)槟[瘤的轉(zhuǎn)移和浸潤(rùn)生長(zhǎng),導(dǎo)致病情的深度惡化而死亡。目前,對(duì)于子宮內(nèi)膜癌的發(fā)病機(jī)理尚未完全清楚,對(duì)于病理分期高、發(fā)生復(fù)發(fā)性轉(zhuǎn)移的患者目前也沒(méi)有確切有效的治療措施,因此,深入研究子宮內(nèi)膜癌的生物學(xué)特點(diǎn),搜尋子宮內(nèi)膜癌相關(guān)標(biāo)記物并分析其與子宮內(nèi)膜癌的分化、轉(zhuǎn)移和侵襲存在的不同差異及其分子機(jī)制,對(duì)提高子宮內(nèi)膜癌病程的判斷預(yù)測(cè)、臨床治療的指導(dǎo)及患者預(yù)后的改善具有重要的意義。微小RNA (micro RNAs, miRNA)是一種由22-25個(gè)核苷酸組成的非編碼單鏈RNAs,目前認(rèn)為在哺乳動(dòng)物體內(nèi),miRNA與靶基因作用,使靶基因的mRNA在轉(zhuǎn)錄后的翻譯過(guò)程中被抑制或直接降解,導(dǎo)致靶基因的蛋白表達(dá)水平降低。通過(guò)這種方式,miRNA參與到包括生長(zhǎng)發(fā)育、造血、器官形成、細(xì)胞增殖、凋亡乃至腫瘤發(fā)生等多種生命活動(dòng)中。據(jù)研究表明,每個(gè)miRNA可以調(diào)控200個(gè)靶基因,同樣一個(gè)靶基因可以被多個(gè)miRNA調(diào)控。生物信息學(xué)則是利用miRNA與靶基因mRNA可以完全或部分互補(bǔ)的原則,通過(guò)各自的堿基序列預(yù)測(cè)出可能與特定miRNA(或mRNA)作用的所有mRNA(或niRNA),以縮小研究范圍,進(jìn)一步通過(guò)實(shí)驗(yàn)驗(yàn)證二者的確切關(guān)系。miRNA-mRNA調(diào)控網(wǎng)絡(luò)定義為在相似生物過(guò)程中的一組miRNAs和一組mRNAs。一個(gè)調(diào)控模塊中包含的miRNAs和靶基因mRNAs都具有高度的關(guān)聯(lián)性,他們被認(rèn)為具有相似的生物功能。目前,較為廣泛應(yīng)用的是MRMs,這種方法就是利用多種異構(gòu)數(shù)據(jù)源miRNA-mRNA堿基互補(bǔ)配對(duì)信息、miRNAs和mRNAs表達(dá)信息來(lái)構(gòu)建miRNA-mRNA調(diào)控網(wǎng)絡(luò),他們他們所發(fā)現(xiàn)的miRNA-mRNA對(duì)具有較高的置信度并且miRNAs與mRNAs的表達(dá)模式高度相關(guān)。因此,通過(guò)挖掘miRNA-mRNA調(diào)控模塊可以在分子層次上了解疾病的生物過(guò)程；其次,可以了解包括癌癥或腫瘤在內(nèi)的疾病生物機(jī)理；另外,還可以為基因診斷和治療提供參考。胞質(zhì)型多聚腺苷酸化原件結(jié)合蛋白(cytoplasmic polyadenylation element binding protein, CPEBs)是一組高度保守的RNA結(jié)合蛋白家族。在其與腫瘤關(guān)系的研究中,以CPEB1和CPEB4研究較為廣泛。但結(jié)合前期工作基礎(chǔ),子宮內(nèi)膜癌高通量測(cè)序提示CPEBs家族中的CPEB1與子宮內(nèi)膜癌的發(fā)生更為密切。已有文獻(xiàn)研究,CPEBs人類(lèi)乳頭瘤病毒(human papilloma virus, HPV)的感染有關(guān)[14]。HPV持久的感染和蛋白質(zhì)E5、E6和E7的表達(dá)是宮頸癌發(fā)生的先決條件,HPVE5、E6、E7 mRNA含有3'不翻譯區(qū)域(3'UTR)特定序列(富含U的胞質(zhì)型聚腺苷酸化原件,CPE區(qū)),它們是CPEB1作用的特異性靶點(diǎn),其多聚腺苷酸化、翻譯受到CPEB1的調(diào)控。CPEB1在HPV陽(yáng)性腫瘤發(fā)展中可能具有重要的調(diào)節(jié)功能,將CPEB mRNA作為生殖器官癌癥的標(biāo)記物應(yīng)該非常有用,因此探究CPEB1在子宮內(nèi)膜癌的發(fā)生中的作用有重要意義。研究目標(biāo)：本研究旨在通過(guò)采用網(wǎng)絡(luò)生物信息學(xué)理論,從子宮內(nèi)膜癌相關(guān)miRNA文獻(xiàn)中挖掘Traits associated miRNA,結(jié)合已知基因功能和miRNA靶基因預(yù)測(cè)數(shù)據(jù)庫(kù),構(gòu)建miRNA和靶基因的綜合調(diào)控關(guān)系網(wǎng)絡(luò),通過(guò)與前期研究工作想結(jié)合并初步驗(yàn)證,篩選出新的與子宮內(nèi)膜癌高度相關(guān)的靶基因CPEB1及以靶基因CPEB1為中心的關(guān)系調(diào)控網(wǎng)絡(luò)；在此基礎(chǔ)上再以獲得性與缺失性功能試驗(yàn)明確所篩選靶基因在子宮內(nèi)膜癌細(xì)胞中的生物學(xué)功能,并初步探求CPEB1在子宮內(nèi)膜癌發(fā)生發(fā)展中可能的分子機(jī)制。方法：使用自由檢索詞,以FACTA工具和Pubmed數(shù)據(jù)庫(kù)進(jìn)行文獻(xiàn)挖掘子宮內(nèi)膜癌相關(guān)miRNAs;4種不同的預(yù)測(cè)方法(TargetScan、miRanda、miRDB和Starbase)預(yù)測(cè)所挖掘出來(lái)每個(gè)miRNA的作用靶基因獲得miRNA-mRNA對(duì)；從miRNA-mRNA對(duì)中選取至少5個(gè)以上miRNA共同靶向的mRNA為子宮內(nèi)膜癌相關(guān)基因,構(gòu)建子宮內(nèi)膜癌的miRNA-mRNA網(wǎng)絡(luò)；利用QRT-PCR法驗(yàn)證調(diào)控網(wǎng)絡(luò)中綜合排名靠前基因mRNA表達(dá)情況,篩選出特異性差異表達(dá)的基因CPEB1為目的基因進(jìn)行深入研究。利用QRT-PCR法檢測(cè)CPEB1在子宮內(nèi)膜癌組織和細(xì)胞中的表達(dá)情況；構(gòu)建CPEB1過(guò)表達(dá)載體,通過(guò)體外轉(zhuǎn)染CPEB1過(guò)表達(dá)載體過(guò)表達(dá)CPEB1,以MTT、克隆形成、流式細(xì)胞術(shù)、劃痕實(shí)驗(yàn)、transwell小室等實(shí)驗(yàn)方式體外檢測(cè)CPEB1對(duì)子宮內(nèi)膜癌細(xì)胞株生物學(xué)行為的影響；構(gòu)建穩(wěn)定過(guò)表達(dá)CPEB1的子宮內(nèi)膜癌細(xì)胞株,以裸鼠成瘤實(shí)驗(yàn)體內(nèi)分析CPEB1的生物學(xué)功能；通過(guò)轉(zhuǎn)染CPEB1過(guò)表達(dá)載體,觀察細(xì)胞形態(tài)變化,應(yīng)用western blot分析細(xì)胞凋亡相關(guān)蛋白以及EMT標(biāo)記關(guān)鍵蛋白分子的表達(dá),初步探討CPEB1參與子宮內(nèi)膜癌細(xì)胞生物學(xué)行為調(diào)控的分子機(jī)制。結(jié)果：1.通過(guò)多步分析,結(jié)合現(xiàn)有近五年來(lái)41篇相關(guān)文獻(xiàn),初步篩選出208個(gè)子宮內(nèi)膜癌相關(guān)miRNA及其靶基因；經(jīng)過(guò)4個(gè)不同數(shù)據(jù)庫(kù)比對(duì)分析,篩選同時(shí)存在于兩個(gè)或兩個(gè)以上數(shù)據(jù)庫(kù),并至少有兩篇研究報(bào)道的miRNA為目標(biāo)miRNA,最終獲得35個(gè)子宮內(nèi)膜癌相關(guān)miRNA。2.通過(guò)4種預(yù)測(cè)方法對(duì)35個(gè)子宮內(nèi)膜癌相關(guān)miRNA進(jìn)行靶基因預(yù)測(cè),獲得110995個(gè)miRNA-mRNA對(duì),去除出現(xiàn)次數(shù)少于3的miRNA-mRNA對(duì),最終獲得由27個(gè)miRNAs和3082個(gè)基因組成的7919個(gè)miRNA-mRNA對(duì),其中至少5個(gè)miRNA同時(shí)靶向的基因457個(gè)。3.分析457個(gè)候選基因的蛋白間的相互作用,有61個(gè)基因至少存在5個(gè)互作蛋白,14個(gè)基因至少有10個(gè)互作蛋白。信號(hào)通路分析這61個(gè)候選基因富集的調(diào)控網(wǎng)絡(luò),大部分通路為癌相關(guān)通路。4.從61個(gè)候選基因中挑選互作蛋白數(shù)量靠前的10個(gè)基因進(jìn)行QRT-PCR實(shí)驗(yàn)驗(yàn)證,有3個(gè)基因表達(dá)失調(diào),其中CDC25A和IGF1R在子宮內(nèi)膜癌中表達(dá)上調(diào),CPEB1表達(dá)下調(diào),CDC25A和IGF1R已有文獻(xiàn)報(bào)道與子宮內(nèi)膜癌密切相關(guān),篩選CPEB1為目標(biāo)基因進(jìn)行功能驗(yàn)證。5.分別檢測(cè)CPEB1在20對(duì)子宮內(nèi)膜癌組織及配對(duì)癌旁組織、3株子宮內(nèi)膜癌細(xì)胞和1株正常子宮內(nèi)膜上皮細(xì)胞的表達(dá)水平,結(jié)果顯示CPEB1在子宮內(nèi)膜癌組織和子宮內(nèi)膜癌細(xì)胞株中表達(dá)顯著下調(diào),提示CPEB1可能作為子宮內(nèi)膜癌發(fā)生的分子標(biāo)記。6. CPEB1體外功能研究結(jié)果顯示：MTT、克隆形成實(shí)驗(yàn)證實(shí)CPEB1能顯著抑制子宮內(nèi)膜癌細(xì)胞的體外增殖能力；流式細(xì)胞凋亡檢測(cè)發(fā)現(xiàn)CPEB1能促進(jìn)子宮內(nèi)膜癌細(xì)胞的凋亡；劃痕實(shí)驗(yàn)證實(shí)CPEB1過(guò)表達(dá)抑制子宮內(nèi)膜癌細(xì)胞的遷移；transwell實(shí)驗(yàn)證實(shí)CPEB1能顯著抑制子宮內(nèi)膜癌細(xì)胞的侵襲能力；小鼠體表皮下成瘤實(shí)驗(yàn)及HE、IHC檢測(cè)證實(shí),CPEB1能顯著抑制腫瘤在體內(nèi)的形成。7. CPEB1在ISK細(xì)胞株中極低表達(dá),CPEB1-siRNA實(shí)驗(yàn)未能成功構(gòu)建CPEB1低表達(dá)干擾載體用于后續(xù)實(shí)驗(yàn)。8.通過(guò)生物信息學(xué)分析及數(shù)據(jù)庫(kù)的預(yù)測(cè),提示CPEB1可能通過(guò)細(xì)胞凋亡通路及EMT相關(guān)通路參與腫瘤疾病的發(fā)生；凋亡相關(guān)分子檢測(cè)結(jié)果顯示,過(guò)表達(dá)CPEB1后,子宮內(nèi)膜癌細(xì)胞ISK中細(xì)胞凋亡相關(guān)分子P53、Bax、Caspase-3表達(dá)上調(diào),Bcl-2表達(dá)下調(diào)；提示CPEB1可能參與P53介導(dǎo)子宮內(nèi)膜癌細(xì)胞凋亡調(diào)控。9.EMT相關(guān)分子檢測(cè)結(jié)果顯示,過(guò)表達(dá)CPEB1后,細(xì)胞發(fā)生上皮化的形態(tài)學(xué)改變。子宮內(nèi)膜癌細(xì)胞ISK中上皮-間質(zhì)轉(zhuǎn)化特征標(biāo)志物和相關(guān)轉(zhuǎn)錄因子E-cadherin、β-catenin的表達(dá)明顯上調(diào),Vimentin、SMA、Snail的表達(dá)顯著下調(diào),Twist不發(fā)生明顯變化,由此證實(shí),CPEB1高表達(dá)促進(jìn)細(xì)胞發(fā)生間質(zhì)-上皮化改變(MET)。結(jié)論1.成功構(gòu)建子宮內(nèi)膜癌特異miRNA-mRNA調(diào)控網(wǎng)絡(luò),篩選CPEB1為子宮內(nèi)膜癌密切相關(guān)基因。2.證實(shí)CPEB1在子宮內(nèi)膜癌組織及細(xì)胞中特異性低表達(dá),CPEB1低表達(dá)可能是子宮內(nèi)膜癌發(fā)生及惡性轉(zhuǎn)移的分子標(biāo)記。3. CPEB1作為抑癌因子,具有抑制子宮內(nèi)膜癌細(xì)胞的生長(zhǎng)和增殖、促進(jìn)子宮內(nèi)膜癌細(xì)胞的凋亡、抑制侵襲和轉(zhuǎn)移,抑制小鼠皮下成瘤腫瘤形成的生物學(xué)功能。4. CPEB1影響子宮內(nèi)膜癌細(xì)胞中細(xì)胞凋亡相關(guān)蛋白P53、Bax、Bcl-2. caspase-3的表達(dá),推測(cè)CPEB1可能通過(guò)P53介導(dǎo)的信號(hào)通路參與子宮內(nèi)膜癌細(xì)胞凋亡的調(diào)控,參與子宮內(nèi)膜癌的發(fā)生。5. CPEB1影響子宮內(nèi)膜癌細(xì)胞中EMT特異標(biāo)記物E-cadherin、Vimentin、 SMA、β-catenin及促EMT轉(zhuǎn)錄因子Snail的表達(dá),推測(cè)CPEB1可能通過(guò)下調(diào)子宮內(nèi)膜癌細(xì)胞EMT轉(zhuǎn)錄因子的表達(dá),促進(jìn)腫瘤發(fā)生MET,抑制腫瘤細(xì)胞的侵襲。
[Abstract]:Background: endometrial carcinoma (EC) is a common malignant tumor of female reproductive system from endometrium epithelial cells. It is one of the three most malignant gynecologic malignancies. The incidence is second only to cervical cancer, which is located in the second place, accounting for about 30% of the cancer of gynecologic cancer. The five year survival rate of the patients with endometrial cancer is not low and the prognosis is good, but it is increasing year by year in our country. It is serious harm to women's health. As a disease that is not clear, 85%-90% is type I endometrium cancer in endometrial cancer, and the research of tumor related diseases is still not very good. The high incidence of endometrial carcinoma is mainly the postmenopausal women, the age of which is 50-60 years old and about 75% of the total incidence of endometrium. In recent years, the incidence and mortality of endometrial cancer are becoming more and more young and rapidly rising in the world. In 2005, 40880 newly discovered endometrial cancer cases were found in the United States, of which 7310 patients died. In 2010, there were 43470 new cases and 7950 deaths. The latest epidemiological data showed that in all new American women's cancers, the incidence of endometrial cancer was significantly increased in 2015, only in line with the incidence of endometrial cancer. The number of breast cancer, lung cancer and colon cancer is the fourth place, and the number of deaths due to endometrial cancer is expected to reach more than 10170. In China, there are no statistical reports on large scale endometrial cancer in China. However, according to the incomplete survey statistics, the trend of the incidence of endometrial cancer in China has been increasing year by year. According to the pathological features of the tumor, endometrial carcinoma is divided into two types: type I estrogen dependence and type II non estrogen dependence. There are obvious differences in the molecular mechanism, morphological characteristics and prognosis of the two. Type I is associated with over stimulation of estrogen, mainly the overgrowth of endometrium. Endometrioid adenocarcinoma, which is based on the development of endometrioid adenocarcinoma, accounts for 80-90% of endometrial carcinoma. Type II pathological types are mainly transparent cell carcinoma, serous papillary carcinoma, mucous adenocarcinoma, and adenosscale carcinoma. The main clinical type of estrogen dependent type I is [9]. Therefore, this study mainly focuses on type I endometrioid adenocarcinoma. Endometrial carcinoma of the endometrium refers to type I endometrioid adenocarcinoma. The early clinical manifestations of endometrial carcinoma are irregular vaginal bleeding, which can help early diagnosis, and 80% of the endometrium cancer patients are localized in the uterus. Therefore, most of the patients with endometrial cancer are compared to other women with reproductive system cancer. The survival rate of the five years is not low and the prognosis is good. The 5 year disease free survival rate of the patients with early endometrial cancer can reach 98%, the total survival rate of 5 years can reach 93%., however, there are still some endometrium cancer patients with low degree of pathological differentiation, high invasion and metastasis rate and poor prognosis, usually because of tumor metastasis and infiltration. At present, the pathogenesis of endometrial cancer is not completely clear. There is no effective and effective treatment for patients with high pathological stages and recurrent metastasis. Therefore, the biological characteristics of endometrial cancer are studied, and the related markers of endometrial cancer are searched and analyzed. The differentiation, metastasis and invasion of endometrial carcinoma have different differences and their molecular mechanisms. It is of great significance to predict the course of endometrial cancer, guide the clinical treatment and improve the prognosis of the patients. RNA (micro RNAs, miRNA) is a non coding single chain RNAs consisting of 22-25 nucleotides, which is currently considered to be feeding. In milk moving objects, miRNA and target genes act to inhibit or directly degrade the target gene mRNA in the post transcriptional translation process, resulting in a decrease in the protein expression level of the target gene. By this way, miRNA participates in a variety of life activities including growth, hematopoiesis, organogenesis, cell proliferation, apoptosis and even tumorigenesis. The study shows that 200 target genes can be regulated by each miRNA, and the same target gene can be regulated by multiple miRNA. Bioinformatics uses the principle that miRNA and target gene mRNA can be fully or partially complementary, and all the mRNA (or niRNA) that may interact with a specific miRNA (or mRNA) can be predicted by their respective base sequences to reduce the scope of the study. Further experiments verify that the exact relationship between the two.MiRNA-mRNA regulatory networks is defined as a high correlation between a group of miRNAs in similar biological processes and the miRNAs and target gene mRNAs contained in a set of mRNAs. regulatory modules, and they are considered to have similar biological functions. At present, MRMs is widely used. The method is to use a variety of heterogeneous data sources miRNA-mRNA base complementary pairing information, miRNAs and mRNAs to construct the miRNA-mRNA regulatory network, they found that the miRNA-mRNA pairs have high confidence and the miRNAs and mRNAs expression patterns are highly correlated. Therefore, the miRNA-mRNA regulation module can be divided by mining the miRNA-mRNA regulation module. The biological processes of disease are understood at the sub level; secondly, we can understand the biological mechanisms of diseases including cancer or tumor; in addition, it can provide reference for genetic diagnosis and treatment. The cytoplasmic polyadenosine acidified original binding protein (cytoplasmic polyadenylation element binding protein, CPEBs) is a highly conserved RNA CPEB1 and CPEB4 are more widely used in the study of the association with the tumor. But combined with the early work basis, high throughput sequencing of endometrial cancer suggests that CPEB1 in the CPEBs family is more closely related to endometrial cancer. The infection of the CPEBs human mammary head tumor virus (human papilloma virus, HPV) has been studied. [14].HPV persistent infection and the expression of protein E5, E6 and E7 are the prerequisites for cervical cancer. HPVE5, E6, and E7 mRNA contain 3'untranslated region (3'UTR) specific sequence (rich in U cytoplasmic polyadenosine acidification original, CPE region). They are the specific targets of the action. The role of CPEB mRNA as a marker of reproductive organ cancer should be very useful in the development of positive tumor. Therefore, it is important to explore the role of CPEB1 in the occurrence of endometrial cancer. Traits associated miRNA was excavated, combined with the known gene function and the miRNA target gene prediction database, the comprehensive regulatory network of miRNA and target genes was constructed. Through the combination of previous research work and preliminary verification, the new target based on endometrial carcinoma was screened by CPEB1 and the target gene CPEB1 as the center. On this basis, the biological function of the screened target gene in endometrial cancer cells is identified by the acquired and missing function test, and the possible molecular mechanism of CPEB1 in the development of endometrial cancer is preliminarily explored. Methods: using free retrieval words, using FACTA tool and Pubmed database to carry out the literature mining in the uterus. Membrane cancer related miRNAs; 4 different prediction methods (TargetScan, miRanda, miRDB and Starbase) predicted that each miRNA target gene was excavated to obtain miRNA-mRNA pairs; at least 5 more miRNA common targets were selected from miRNA-mRNA pairs for endometrial cancer related basis, and the miRNA-mRNA network of endometrial cancer was constructed; Q was used for Q. The RT-PCR method was used to verify the expression of mRNA in the regulatory network, and the specific gene CPEB1 was screened out for the target gene. QRT-PCR was used to detect the expression of CPEB1 in endometrial carcinoma tissues and cells; to construct CPEB1 overexpressed carrier and transfect CPEB1 overexpression vector in vitro The effect of CPEB1 on the biological behavior of endometrial carcinoma cell lines was detected by MTT, cloned formation, flow cytometry, scratching experiment, and Transwell chamber. The biological function of the endometrial carcinoma cell line that stably overexpressed CPEB1 was constructed, and the biological function of CPEB1 in nude mice was analyzed in vivo of the tumor in nude mice; through transfection of CPEB1 over the table of CPEB1 The molecular mechanism of CPEB1 involvement in the regulation of biological behavior of endometrial cancer cells was preliminarily investigated by Western blot analysis of cell morphological changes. The molecular mechanism of CPEB1 involvement in the regulation of biological behavior of endometrial cancer cells was preliminarily discussed. Results: 1. through multistep analysis, 41 related documents have been screened in recent five years, and a preliminary screening was made. Endometrial carcinoma associated miRNA and its target genes were screened by 4 different database comparisons and at least two or more databases were screened at the same time, and at least two reported miRNA were targeted miRNA, and 35 endometrial cancer related miRNA.2. was finally obtained by 4 pretests for 35 endometrial cancer related miRNA. Target gene prediction, obtained 110995 miRNA-mRNA pairs, the removal of less than 3 of the miRNA-mRNA pair, and finally got 7919 miRNA-mRNA pairs of 27 miRNAs and 3082 genes, of which at least 5 miRNA targeted gene 457.3. analysis of the protein interaction between 457 candidate genes, 61 genes at least 5 interacts. Protein, 14 genes have at least 10 interacting proteins. The signaling pathway analyses the regulatory network of the 61 candidate genes, and most of the pathways are the cancer related pathway.4. from 61 candidate genes to select 10 genes from the 61 candidate genes to perform the QRT-PCR test, and 3 genes are dysfunctional, of which CDC25A and IGF1R are in endometrial cancer. The expression of CPEB1, CDC25A and IGF1R were closely related to endometrial cancer, and CPEB1 was selected as the target gene for functional verification..5. was used to detect CPEB1 in 20 endometrial and paracancerous tissues, 3 endometrial cancer cells and 1 normal endometrium epithelial cells. The expression of CPEB1 in endometrial carcinoma and endometrial carcinoma cell lines showed a significant downregulation, suggesting that CPEB1 may be a molecular marker of endometrial carcinoma in vitro. The results of the function of.6. CPEB1 in vitro showed: MTT, the clone formation experiment confirmed that CPEB1 could significantly inhibit the proliferation ability of endometrial carcinoma cells in vitro; flow cytometry It was found that CPEB1 could promote the apoptosis of endometrial cancer cells, and the scratch test confirmed that CPEB1 overexpression inhibited the migration of endometrial cancer cells, and the Transwell experiment confirmed that CPEB1 could significantly inhibit the invasion ability of endometrial cancer cells, the tumor formation experiment under the epidermis of the mice and the HE, IHC test evidence, CPEB1 could significantly inhibit the form of the tumor in the body. The expression of.7. CPEB1 was very low in ISK cell lines, and the CPEB1-siRNA experiment failed to construct CPEB1 low expression interference carrier for the follow-up experiment.8. through bioinformatics analysis and the prediction of database, suggesting that CPEB1 may participate in the occurrence of tumor disease through the apoptosis pathway and EMT related pathway; apoptosis related molecular detection results show that After overexpression of CPEB1, the apoptosis related molecules P53, Bax, Caspase-3, and Bcl-2 expression are up regulated in endometrial carcinoma cells, suggesting that CPEB1 may participate in the regulation of P53 mediated apoptosis of endometrial carcinoma cells and the results of.9.EMT related molecular detection show that after overexpression of CPEB1, the morphological changes of epithelial cells occur. Endometrial carcinoma is fine. The expression of epithelial mesenchymal transition in cell ISK and related transcription factor E-cadherin, the expression of beta -catenin was obviously up-regulated, the expression of Vimentin, SMA, Snail decreased significantly, and Twist did not change obviously. Thus, the high expression of CPEB1 promoted the change of cytoplasm epithelialization of cells (MET). Conclusion 1. successfully constructed the specific miRNA-mRNA of endometrial carcinoma. Regulatory network, screening CPEB1 for endometrial cancer closely related gene.2. confirmed that CPEB1 in endometrial cancer tissues and cells of low expression, CPE
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.33

【相似文獻(xiàn)】

相關(guān)會(huì)議論文 前1條

1 周鵬;彭學(xué)齡;徐唯一;羅鎮(zhèn)華;蔣嵩山;;全基因組范圍miRNA篩選揭示未被預(yù)測(cè)到的miRNA-mRNA相互作用[A];中國(guó)的遺傳學(xué)研究——遺傳學(xué)進(jìn)步推動(dòng)中國(guó)西部經(jīng)濟(jì)與社會(huì)發(fā)展——2011年中國(guó)遺傳學(xué)會(huì)大會(huì)論文摘要匯編[C];2011年

相關(guān)博士學(xué)位論文 前1條

1 熊漢真;Ⅰ型子宮內(nèi)膜腺癌miRNA-mRNA調(diào)控網(wǎng)絡(luò)分析及CPEB1對(duì)其生物學(xué)行為的影響[D];南方醫(yī)科大學(xué);2016年

相關(guān)碩士學(xué)位論文 前2條

1 張俊鵬;基于概率話題模型的功能性miRNA-mRNA調(diào)控模塊識(shí)別[D];昆明理工大學(xué);2012年

2 楊梅;2型糖尿病GWAS關(guān)聯(lián)SNP對(duì)miRNA-mRNA互作的影響分析[D];華中師范大學(xué);2015年

，

本文編號(hào)：1904405