本文選題：子宮內膜癌 + microRNA�。� 參考：《廣西醫(yī)科大學》2015年博士論文

【摘要】：子宮內膜癌(endometrial carcinoma, EC)是最常見的婦科惡性腫瘤之一,嚴重威脅婦女健康。近年由于環(huán)境、平均壽命延長、激素替代療法應用等各種因素的影響,其發(fā)病率逐年上升。美國癌癥協(xié)會統(tǒng)計在2012年全球大概有47130例新發(fā)子宮內膜癌患者,已經躍居發(fā)達國家女性生殖道惡性腫瘤首位。盡管目前對子宮內膜癌的治療方法和技術不斷發(fā)展,但對晚期和復發(fā)的子宮內膜癌的治療仍為棘手。進一步研究和尋找子宮內膜癌的新的治療手段尤為重要。國內外研究證實腫瘤發(fā)生發(fā)展是一個復雜的分子調控失控過程,與抑癌基失活、癌基因的激活從而導致細胞生長失控,細胞基質間黏附及細胞信號傳導異常密切聯(lián)系。因此,探索子宮內膜癌發(fā)生的分子學基礎,尋找安全可靠的切入點抑制子宮內膜癌細胞生長,轉移,對預防子宮內膜癌的發(fā)生和改善子宮內膜癌患者的預后,保護我國婦女健康具有重大意義。MicroRNA(miRN A)是近年來發(fā)現(xiàn)的一類保守的長度為19～24個堿基的非編碼RNA分子,廣泛存在真核生物體內,其通過與靶基因mRNA3’非翻譯區(qū)(3’UTR)相對應的靶序列完全或不完全的互補結合,抑制或促進mRNA降解,調節(jié)基因,這些僅占人類基因1%的miRNA分子,卻調控著人類三分之一以上基因的表達、修飾、轉錄和翻譯的過程,對細胞增殖、分化及凋亡等生物學功能起著重要的調控作用。miRNA是腫瘤研究領域中的熱點,其包括miRNA表達譜篩選,分子結構,調控機制,靶基因預測等研究。越來越多的研究證實,niRNA在多種惡性腫瘤中存在表達異常,在腫瘤中高表達的niRNA可能發(fā)揮癌基因的作用,而低表達的miRNA發(fā)揮抑癌基因作用。miRNA的發(fā)現(xiàn)可能成為極有應用價值的分子標志物,為惡性腫瘤的早期診斷和預后評估提供新方向。miR-944是目前學者關注的新焦點,目前國內外對關于miR-944功能的報道還比較少,初步研究發(fā)現(xiàn)：miR-944在子宮內膜癌、宮頸癌及胰腺癌等惡性腫瘤中表達顯著增高,我們推測其在腫瘤中發(fā)揮癌基因的作用。但迄今為止,對子宮內膜癌組織進行差異表達miRNA的篩選及分析研究甚少。本課題旨在闡明子宮內膜癌組織中的miRNA表達譜及miR-944對子宮內膜癌生物學行為影響及調控作用的機制研究。研究內容包括以下四部分：第一部分 子宮內膜癌組織中差異表達miRNA的篩選研究目的：篩選出子宮內膜癌組織中的差異表達的miRNA分子,獲得子宮內膜癌miRNA表達譜,尋找新的診斷標志物。方法：收集3例子宮內膜癌患者的癌變內膜組織和3例正常對照組子宮內膜組織,提取RNA,應用丹麥Exiqon公司的miRCURYTM LNA miRNA芯片,篩選出子宮內膜癌組織中的差異表達的miRNA分子,采用實時定量逆轉錄聚合酶鏈反應(qRT-PCR)在樣本中進行驗證。結果：1.3例癌組織和3例正常內膜組織的總RNA抽取及質檢,結果顯示,所提取的總RNA的電泳可見帶28S和18s條帶,260／A280的值都介于1.8-2.1之間,檢測RNA檢測結果符合雜交芯片實驗要求。2. miRNA芯片結果顯示：子宮內膜癌組織與正常子宮內膜組織存在有顯著差異表達的miRNA:有14個miRNA表達上調,6個miRNA表達下調。3.采用qRT-PCR方法對上調的miR-944、miR-373及下調的miR-548、miR-885進行驗證,結果顯示：子宮內膜癌中miR-944、miR-373上調的倍數(shù)為3.13倍和4.2倍,1niR-548、miR-885下調倍數(shù)為2.08倍及3.84倍,與芯片結果比較,趨勢一致。結論miRNA表達譜芯片可用于分析子宮內膜癌組織miRNA表達譜；通過miRCURYTMLNA miRNA芯片篩選結合qRT-PCR驗證,共得到14個顯著上調6個顯著下調的miRNA,為下一步開展子宮內膜癌組織niRNA的研究及功能學研究提供實驗基礎。第二部分 子宮內膜癌組織中miR-944表達及其與臨床病理特征的關系目的：探討miR-944在子宮內膜癌組織中的表達,并分析其與臨床病理特征的關系。方法：收集2012年3月—2014年4月在廣西醫(yī)科大學第一附屬醫(yī)院進行手術治療的子宮內膜癌患者的癌變內膜組織40例,收集同期因子宮肌瘤等良性病變行子宮切除術的正常子宮內膜組織25例作為對照。采用qRT-PC R方法進行miR-944表達水平的相對定量檢測,并分析其與患者臨床病理特征的關系。結果：1.miR-944在子宮內膜癌組織及正常子宮內膜組織中的表達子宮內膜癌組miR-944的表達水平高于及正常內膜組(P0.05)。2.miR-944與臨床病理特征的關系miRNA-944的表達與組織學分級、病理類型、淋巴轉移、肌層浸潤深度及FIGO分期因素相關,均有統(tǒng)計學意義(P0.05),與年齡無相關性。結論：miR-944在子宮內膜癌組織中高表達,提示miR-944可能參與子宮內膜癌發(fā)生及發(fā)展；miR-944的表達與組織學分級、病理類型、淋巴轉移、肌層浸潤深度及FIGO分期因素相關,可能可以預測子宮內膜癌的惡性程度及不良預后。第三部分 miR-944對子宮內膜癌細胞生物學行為的影響目的：探討miR-944對子宮內膜癌Ishikawa細胞和KLE細胞生物學行為的影響。方法：人工合成miR-944的模擬物mimics及抑制物inhibitor,利用陽離子脂質體法,分別轉染至人子宮內膜癌Ishikawa細胞和KLE細胞中。設置陰性對照組及空白對照組。采用Cell Counting Kit-8(CCK-8)法對比轉染前后細胞增殖生長能力的區(qū)別；流式細胞學對比轉染前后細胞凋亡情況的區(qū)別；Transwell小室法對比轉染前后細胞遷移和侵襲能力的區(qū)別。結果：1.細胞轉染效果熒光倒置顯微鏡觀察轉染后的miR-944 mimics的轉染效率,通過熒光鏡下視野與光鏡下視野對比,Ishikawa細胞和KEL細胞的轉染效率大約為85%。2. miR-944-mimics及miR-944-inhibitor(?)子宮內膜癌Ishikawa細胞KLE細胞生物學行為的影響。(1)CCK8法檢測細胞增殖實驗分別在(24h、48h、72h、96h)四個時間點行CCK8檢測Ishikawa和KLE細胞的OD值,結果顯示,相對于對照組細胞,轉染了miR-944mimics的Ishikawa細胞和KLE細胞增殖活性均增強,轉染了niR-944inhibitor的Ishikawa細胞和KLE細胞增殖活性均降低(P0.05)。(2)流式細胞術檢測細胞凋亡對miR-944轉染48h后的Ishikawa細胞進行流式細胞術細胞凋亡分析,結果顯示,B組、NC組、miR-944inhibitor組和miR-944mimics組凋亡率分別為0.58±0.06%、0.66±0.07%、11.74±0.19%、0.68±0.03%, miR-944inhibitor組凋亡率明顯高于B組(P0.05),niR-944mimics組凋亡率與B組差別無統(tǒng)計學意義(P0.05)。對miR-944轉染48h后的KLE細胞進行流式細胞術細胞凋亡分析,結果顯示,B組、NC組、miR-944 inhibitor組和miR-944mimics組凋亡率分別為2.55±0.23%、2.53±0.10%、5.13±0.14%、2.99±0.18%,miR-944inhibitor凋亡率明顯高于B組(P0.05),miR-944mimics組凋亡率與B組差別無統(tǒng)計學意義(P0.05)。(3)細胞侵襲實驗檢測轉染48h后各組KLE細胞遷移能力,對穿膜細胞進行拍照和計數(shù),結果顯示,轉染miR-944 mimics的KLE細胞穿膜數(shù)量較B組明顯增多(P0.05)；轉染miR-944 inhibitor的KLE細胞穿膜數(shù)量較B組明顯減少(P0.05)。Ishikawa細胞過膜后鋪展性不好,拍照后無法計數(shù),CCK8方法檢測過膜細胞,結果顯示：相對于對照組細胞,轉染了miR-944 mimics的 Ishikawa細胞OD值高,間接反映了mimics組的穿膜數(shù)量多；轉染miR-944 inhibitor 的 Ishikawa細胞OD低,間接反映了inhibitor組的穿膜數(shù)量少(P0.05)。結論：miR-944可促進子宮內膜癌細胞的增殖,侵襲與轉移能力,降低miR-944水平可促進細胞凋亡,表明miR-944可能發(fā)揮癌基因的作用參與子宮內膜癌的發(fā)生發(fā)展。第四部分 miR-944對子宮內膜癌細胞生物學行為調控的靶基因預測及鑒定目的：預測并實驗證實miR-944的靶基因,初步探討miR-944的生物學機制。方法：登陸1nicroRN A庫及靶基因預測的網站,在線檢測或下載靶基因預測軟件,綜合運用TargetScan(http://genes.mit.edu/targetscan)、Mirbase (http://mirbase.org)、miRanda (http://www.microrna.org)生物信息篩選miR-944最可能的靶基因。Western blot (WB)對比轉染前后細胞表達靶基因的區(qū)別。構建最可能的靶基因的3’UTR雙熒光素酶報告載體,檢測熒光素的活性以期在基因水平上驗證靶基因上存在miR-944的作用靶點。結果：1. 預測miR-944的靶基因通過預測軟件miRanda、Mirbase及TargetScan預測miR-944的靶基因,三個軟件交集的niRNA-944靶基因共70個；登陸基因庫,查詢以上靶基因功能,尤其注意與惡性腫瘤侵襲轉移密切相關的功能基因。最后選擇非受體型蛋白酪氨酸磷酸酶14(protein tyrosine phosphatase non-receptor type 14,PTPN14)作為miR-944的可能靶基因來進一步研究。應用Targetscan預測miR-944與PTPN14基因的結合位點。2.蛋白水平驗證PTPN14是否為miR-944的靶基因miR-944mimics組的KLE細胞的PTPN14蛋白表達量與B組比較下降,差異有統(tǒng)計學意義(P0.05)；niR-944inhibitor組則高于B組,差異有統(tǒng)計學意義(P0.05)表明miR-944模擬物能降低KLE細胞中PTPN14蛋白的表達,B組與NC組比較無統(tǒng)計學意義。在Ishikawa細胞中,miR-944mimics組,miR-944 inhibitor組的PTPN14蛋白表達量與B組均無差異(P0.05)。3. miR-944與PTPN14的結合結合靶點驗證將niR-944mimics與連接了PTPN14基因3'-UTR區(qū)的pmirGLO載體共轉染KLE細胞,實驗設立陽性對照組及陰性對照組,重組報告質粒的熒光素酶活性,結果顯示與對照組比較,實驗組的熒光素酶活性顯著下調,差別有統(tǒng)計學意義(P0.05)結論：PTPN14是miR-944是其中一個靶基因,抑制PTPN14的表達,可能是miR-944促進子宮內膜癌細胞增殖及侵襲轉移的作用機制之一。
[Abstract]:Endometrial carcinoma (EC) is one of the most common gynecologic malignancies. It is a serious threat to women's health. In recent years, the incidence of the cancer is increasing year by year because of the influence of environment, prolonged life expectancy, and hormone replacement therapy. The American Cancer Association combined with 47130 new endometrium cancers worldwide in 2012. Patients have been in the first place in women's reproductive tract cancer in developed countries. Although the treatment and techniques for endometrial cancer are developing continuously, the treatment of advanced and recurrent endometrial cancer is still difficult. Further research and search for new treatment hand Duan You for endometrial cancer are important. Domestic and foreign studies have confirmed the occurrence of tumor. Development is a complex process of uncontrolled molecular regulation, which is associated with the inactivation of tumor suppressor base, the activation of the oncogene and the close relationship between cell growth out of control, cell matrix adhesion and abnormal cell signal transduction. Shift, to prevent endometrial cancer and improve the prognosis of endometrial cancer patients, the protection of the health of women in China is of great significance.MicroRNA (miRN A) is a class of conserved non coded RNA molecules with 19~24 bases in length. There is a wide range of eukaryotes, and it passes through the target gene mRNA3 'non translation zone (3' UTR). A complete or incomplete combination of corresponding target sequences, inhibiting or promoting mRNA degradation and regulating genes, which only occupy 1% of the human gene miRNA, regulate the expression, modification, transcription and translation of more than 1/3 of the human genes, and play an important role in regulating biological functions such as cell proliferation, differentiation and apoptosis,.Mi RNA is a hot spot in the field of cancer research, which includes the screening of miRNA expression spectrum, molecular structure, regulation mechanism and target gene prediction. More and more studies have proved that niRNA has abnormal expression in a variety of malignant tumors, and the high expression of niRNA in the tumor may play the role of cancer based cause, and the low expression miRNA plays the role of tumor suppressor gene. The discovery of miRNA may be a highly valuable molecular marker, providing new direction for the early diagnosis and prognosis evaluation of malignant tumor..miR-944 is a new focus of attention at present. There are few reports about miR-944 function at home and abroad. The preliminary study found that miR-944 is in endometriosis, cervix and pancreatic cancer. There is a significant increase in the expression of the tumor in the tumor, we speculate that it plays the role of the oncogene in the tumor. But so far, the screening and analysis of differential expression of miRNA in endometrial carcinoma tissue is very few. The purpose of this study is to clarify the expression of miRNA in endometrial carcinoma and the effect of miR-944 on the biological behavior of endometrial carcinoma and its regulation and control. Research on the mechanism of action. The study includes the following four parts: the first part: the screening of differential expression of miRNA in endometrial carcinoma: screening the differentially expressed miRNA molecules in endometrial carcinoma, obtaining the miRNA expression profile of endometrial carcinoma and finding new diagnostic markers. Methods: 3 cases of endometrial carcinoma were collected. The endometrial tissue of the patients and the endometrium of 3 normal controls, RNA, and the miRCURYTM LNA miRNA chip of Danish Exiqon company were used to screen the differentially expressed miRNA molecules in endometrial carcinoma tissue, and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the samples. Results: 1.3 cases of cancer tissue were found. The total RNA extraction and quality examination of 3 cases of normal endometrium showed that the extracted total RNA electrophoresis showed 28S and 18S bands, and the values of 260 / A280 were between 1.8-2.1. The detection results of RNA conformed to the requirements of the hybridization chip experiment. The results of.2. miRNA chip showed that there was a significant difference between endometrial carcinoma tissue and normal endometrium. The differential expression of miRNA: was up to 14 miRNA expressions, and 6 miRNA expression down-regulation.3. was verified by qRT-PCR method for up regulated miR-944, miR-373 and down regulated miR-548 and miR-885. The results showed that the multiplier of miR-944, miR-373 up-regulated in endometrial carcinoma was 3.13 times and 4.2 times, 1niR-548, 2.08 times and 3.84 times the down-regulation of 1niR-548. The results were the same. Conclusion the miRNA expression chip can be used to analyze the miRNA expression profile of endometrial carcinoma tissue. Through the miRCURYTMLNA miRNA chip screening and qRT-PCR verification, 14 significantly up regulation 6 down regulated miRNA are obtained, which can provide experimental basis for the next step in the study and functional study of endometrial carcinoma group niRNA. Second part of the expression of miR-944 in endometrial carcinoma and its relationship with the clinicopathological features: To explore the expression of miR-944 in endometrial carcinoma and to analyze its relationship with the clinicopathological features. Methods: to collect the uterus from March 2012 to April 2014 at the First Affiliated Hospital of Guangxi Medical University. 40 cases of endometrial carcinoma in endometrial carcinoma, 25 cases of normal endometrium with hysterectomy and hysterectomy were collected for the same period as control. The relative quantitative detection of miR-944 expression was carried out by qRT-PC R method, and the relationship with the patients' clinicopathological characteristics was analyzed. Results: 1.miR-944 was in endometrial carcinoma. The expression level of endometrial carcinoma group miR-944 in tissue and normal endometrium was higher than that of normal endometrium (P0.05).2.miR-944 and clinicopathological features. The expression of miRNA-944 was related to histological classification, pathological type, lymphatic metastasis, depth of myometrium infiltration and FIGO staging, and was statistically significant (P0.05). Conclusion: miR-944 is highly expressed in endometrial carcinoma, suggesting that miR-944 may be involved in the occurrence and development of endometrial carcinoma, and the expression of miR-944 is related to histological classification, pathological type, lymphatic metastasis, depth of myometrium infiltration and FIGO staging, which may predict the malignancy and poor prognosis of endometrial carcinoma. The effect of the three part of miR-944 on the biological behavior of endometrial carcinoma cells: To explore the effect of miR-944 on the biological behavior of endometrial carcinoma Ishikawa cells and KLE cells. Methods: artificial synthesis of miR-944 simulants, mimics and inhibitor inhibitor, were transfected to human endometrial carcinoma Ishikawa cells by cationic liposome method. And KLE cells. Set negative control group and blank control group. Cell Counting Kit-8 (CCK-8) method was used to compare the proliferation and growth ability of cells before and after transfection; flow cytology compared the difference of cell apoptosis before and after transfection; Transwell cell method compared the difference of cell migration and invasion before and after transfection. Results: 1. cells Transfection effect with fluorescence inverted microscope to observe transfection efficiency of miR-944 mimics after transfection, the transfection efficiency of Ishikawa cells and KEL cells is about 85%.2. miR-944-mimics and miR-944-inhibitor (?) Ishikawa cell KLE cell biological behavior of 85%.2. miR-944-mimics and miR-944-inhibitor (?) endometrial carcinoma. (1) CCK8 method CCK8 detection of Ishikawa and KLE cells at four time points (24h, 48h, 72h, 96h) detected the OD values of Ishikawa and KLE cells respectively. The results showed that the proliferation activity of Ishikawa cells and KLE cells transfected with miR-944mimics was enhanced compared with that of the control group, and both niR-944inhibitor Ishikawa cells and cell proliferation activities were reduced. (2) flow cytometry was used to detect apoptosis in Ishikawa cells transfected with 48h by flow cytometry. The apoptosis rate of B group, NC group, miR-944inhibitor group and miR-944mimics group was 0.58 + 0.06%, 0.66 + 0.07%, 11.74 + 0.19% and 0.68 + 0.03%, respectively. The apoptosis rate of miR-944inhibitor group was significantly higher than that of the B group (P0.05). There was no significant difference in apoptosis rate between group niR-944mimics and group B (P0.05). The apoptosis of KLE cells after miR-944 transfected with 48h showed that the apoptosis rate of B group, NC group, miR-944 inhibitor group and miR-944mimics group was 2.55 + 0.23%, 2.53 + 0.10%, 5.13 + 0.14%, 2.99 + 0.18%, and the apoptotic rate of miR-944inhibitor was obvious Higher than group B (P0.05), there was no significant difference in the apoptosis rate between group miR-944mimics and group B (P0.05). (3) cell invasion test was used to detect the migration ability of KLE cells in each group after transfection of 48h and to take pictures and count of membrane cells. The results showed that the number of membrane transfected on miR-944 mimics KLE cells was significantly higher than that of B group (P0.05). The number of LE cells was significantly less than that in the B group (P0.05), the spread of.Ishikawa cells was not good, and the number of membrane cells could not be counted after taking pictures. CCK8 method detected the membrane cells. The result showed that compared with the control group, the Ishikawa cells transfected with miR-944 mimics had high OD value, which indirectly reflected the number of membrane in the mimics group, and the transfection of miR-944 inhibitor. The Ishikawa cell OD is low, which indirectly reflects the small number of membrane in the inhibitor group (P0.05). Conclusion: miR-944 can promote the proliferation, invasion and metastasis of endometrial cancer cells, and reduce the level of miR-944 to promote cell apoptosis. It is suggested that miR-944 may play the role of oncogene in the development of endometrial carcinoma. The fourth part of miR-944 against the child. The target gene prediction and identification of the biological behavior regulation of endometrial carcinoma cells: predict and verify the target gene of miR-944 and preliminarily discuss the biological mechanism of miR-944. Methods: landing on the 1nicroRN A library and the site of target gene prediction, on-line detection or downloading target gene prediction soft parts, and using TargetScan (http://genes.mit.edu/ta) Rgetscan), Mirbase (http://mirbase.org), miRanda (http://www.microrna.org) biological information screening miR-944 the most likely target gene.Western blot (WB) to compare the difference between the target gene and the target gene before and after the transfection. The 3 'UTR double luciferase reporter vector of the most likely target gene is constructed to detect the activity of fluorescein at the gene level. The target genes of miR-944 were tested on the target gene. Results: 1. the target gene for predicting miR-944 was predicted by the software miRanda, Mirbase and TargetScan for the prediction of the target genes of miR-944, and 70 niRNA-944 target genes of the three software intersections; the landfall gene pool was used to query the function of the target gene, especially closely related to the invasion and metastasis of malignant tumor. Finally, we chose non receptor type protein tyrosine phosphatase 14 (protein tyrosine phosphatase non-receptor type 14, PTPN14) as a possible target gene for miR-944 to further study. Using Targetscan to predict the.2. protein of the binding site of miR-944 and PTPN14 genes to verify that PTPN14 is the target gene. The expression of PTPN14 protein in KLE cells in group imics was lower than that in group B (P0.05), and in group niR-944inhibitor, the difference was statistically significant (P0.05) showed that the miR-944 mimic could reduce the expression of PTPN14 protein in KLE cells, and there was no statistical significance in B group. In group miR-944, the expression of PTPN14 protein in group inhibitor was not different from that of B group (P0.05).3. miR-944 and PTPN14 binding target verifying that niR-944mimics was co transfected with pmirGLO carrier of 3'-UTR region of PTPN14 gene, and the experiment set up positive control group and negative control group. Compared with the control group, the luciferase activity of the experimental group was significantly reduced, and the difference was statistically significant (P0.05) conclusion: PTPN14 was one of the target genes, which inhibited the expression of PTPN14, which may be one of the mechanisms of miR-944 to promote the proliferation and invasion and metastasis of endometrial carcinoma cells.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.33

【參考文獻】

相關期刊論文 前10條

1 陳金明,李偉華,王家頓,馮永東,吳劍宏,龔建平;結腸癌細胞凋亡與增殖的平衡及其與預后的關系[J];癌癥;2005年05期

2 陳玉芬;;子宮內膜癌患者血清中miR-155的表達及其臨床意義研究[J];中國醫(yī)藥導刊;2014年01期

3 薛紀森;張乾;李曉琳;胡燕;楊孝軍;顏笑健;鄭飛云;;子宮內膜癌Ⅰ型和Ⅱ型miRNA表達譜差異的分析[J];實用醫(yī)學雜志;2011年22期

4 劉潛;付潔;宋海峰;;微小RNA分析技術研究進展[J];生物技術通訊;2013年05期

5 何中慧;徐紅;牛菲菲;;脾酪氨酸激酶在子宮內膜癌中的表達及意義[J];實用醫(yī)學雜志;2014年11期

6 劉立鵬;于瀟華;王曉春;郭小芳;田智;龍昱;粟敏;羅sベ，

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