【摘要】：在我國，子宮內膜癌是女性生殖道最常見的惡性腫瘤之一，且其發(fā)病率逐年持續(xù)上升。約25%的子宮內膜癌病例在確診時已屬晚期，對化療和內分泌治療的客觀反應率很低，預后不佳。然而,迄今我們對子宮內膜癌發(fā)生、進展及侵襲轉移的分子機制尚不清楚。 據(jù)報道,同源盒轉錄基因EMX2在雌性生殖系統(tǒng)發(fā)育中起關鍵作用，Emx2-/-雌性小鼠的生殖道（子宮、宮頸、陰道等）完全缺如。在絕經(jīng)前子宮內膜中，，EMX2的蛋白表達量隨月經(jīng)周期發(fā)生規(guī)律性改變，而絕經(jīng)后子宮內膜中EMX2呈持續(xù)性高表達；同時，體外實驗證實EMX2可拮抗正常內膜上皮細胞的過度增殖。課題組預實驗發(fā)現(xiàn)約30%（10/30）的子宮內膜癌組織中EMX2表達顯著下降，提示EMX2基因失活可能參與了子宮內膜癌的發(fā)生發(fā)展等過程。 基于以上發(fā)現(xiàn)，本課題擬從以下四個方面對子宮內膜癌中EMX2基因的生物學功能及其作用機制進行探討：（1）檢測組織和細胞系EMX2mRNA和蛋白的表達水平,分析其與患者各臨床病理參數(shù)之間的相關性；（2）應用甲基化特異性PCR(MSP)及DNA測序等技術檢測子宮內膜癌中EMX2基因啟動子區(qū)高甲基化的發(fā)生頻率，確定DNA甲基化是否可導致EMX2表達下降，并在相應的子宮內膜癌細胞中進行驗證；（3）在子宮內膜癌細胞中分別上、下調EMX2的表達，采用MTT、克隆形成、wound healing、Transwell、流式細胞術、real time PCR和western blot等方法檢測EMX2基因對細胞增殖、遷移、侵襲等生物學行為的影響；建立子宮內膜癌裸鼠荷瘤模型，體內驗證EMX2對子宮內膜癌的抑制作用（；4）采用real time PCR、western blot檢測EMX2對Wnt信號通路的影響，應用Wnt信號通路抑制劑(XAV-939)和激動劑(LiCl)驗證Wnt信號是否為EMX2基因抗腫瘤作用的關鍵下游通路。 第一部分EMX2在子宮內膜癌組織和細胞系中的表達目的：檢測人子宮內膜癌組織和永生化子宮內膜癌細胞系中EMX2的表達水平，探討EMX2表達與患者臨床病理參數(shù)之間的相關性。 方法：應用免疫組織化學方法技術檢測正常內膜組織（n=25）、子宮內膜癌組織（n=122）中EMX2蛋白的表達和定位。應用real time PCR、western blot檢測子宮內膜癌細胞系中EMX2mRNA和蛋白的表達水平。應用SPSS軟件分析EMX2表達與患者臨床病理參數(shù)之間的相關性。 結果： (1)與正常內膜相比，48.4%（59/122）的子宮內膜癌組織中EMX2蛋白表達下降（P 0.001）。EMX2表達下降與腫瘤分期(P=0.023)、分級(P=0.016)、深肌層浸潤(P=0.04)密切相關，與年齡、淋巴脈管間隙浸潤、淋巴結侵犯以及雌孕激素受體狀態(tài)無相關性。 (2)在正常內膜組織中，EMX2蛋白主要定位于細胞核，部分可伴細胞漿同時著色。在子宮內膜癌組織中未見EMX2蛋白著色部位的異常。 (3) KLE、Ishikawa細胞中EMX2表達較高，RL95-2、AN3CA、SPEC-2細胞中EMX2表達極低。結論：EMX2在子宮內膜癌組織中頻發(fā)失活，且與腫瘤惡性程度明顯相關，提示EMX2失活可能參與了子宮內膜癌的發(fā)生、進展。 第二部分子宮內膜癌中EMX2基因失活機制 目的：探討人子宮內膜癌中EMX2基因失活的機制。 方法：應用甲基化特異性PCR(MSP)及測序技術檢測子宮內膜癌細胞系（n=4）、正常內膜（n=22）和子宮內膜癌組織（n=79）中EMX2基因啟動子區(qū)發(fā)生甲基化的頻率；應用甲基化逆轉藥物5-AZA-dC處理子宮內膜癌細胞，采用real time PCR、western blot分別檢測細胞內EMX2mRNA和蛋白水平的改變。 結果： (1)在正常內膜和子宮內膜癌組織中，EMX2基因啟動子高甲基化的發(fā)生率分別為4.5%（1/22）和39.2%（32/79，P 0.001），且高甲基化與EMX2表達失活明顯相關(P 0.001)。 (2)僅RL95-2細胞中存在EMX2啟動子高甲基化；經(jīng)1μM5-AZA-dC處理12小時后，RL95-2細胞中EMX2mRNA和蛋白表達均明顯上升，而其他3種細胞中EMX2表達無明顯變化。 結論：啟動子高甲基化是導致子宮內膜癌中EMX2基因失活的一個重要因素，同時也提示EMX2可能是一個抑癌基因。 第三部分EMX2基因對子宮內膜癌細胞生物學行為的影響目的：探討EMX2對子宮內膜癌細胞增殖、凋亡、周期、遷移、侵襲和成瘤能力的影響。方法：使用pcDNA3.1-EMX2、siRNA-EMX2及它們的陰性對照對RL95-2和KLE細胞分別進行瞬時轉染，應用MTT、克隆形成實驗檢測細胞增殖能力；應用流式細胞術檢測細胞凋亡、周期的改變；應用劃痕實驗檢測細胞遷移能力；應用Transwell小室實驗檢測細胞侵襲能力。應用pcDNA3.1-EMX2轉染RL95-2細胞并用G418篩選、構建EMX2穩(wěn)定高表達細胞株，應用該細胞株在裸鼠體內驗證EMX2對腫瘤形成的影響。 結果： (1)轉染效果檢測：瞬時轉染pcDNA3.1-EMX2質粒后，RL95-2細胞中EMX2mRNA和蛋白水平均顯著升高；瞬時轉染siRNA-EMX2后，KLE細胞中EMX2mRNA和蛋白水平下降超過75%。 (2)上調EMX2表達后，RL95-2細胞的增殖(P=0.002, MTT; P=0.03,克隆形成)、遷移(P=0.018)、侵襲(P=0.039)能力受到明顯抑制；同時EMX2可將RL95-2細胞阻滯在G1期；但EMX2并不增加RL95-2細胞的凋亡率。而下調EMX2表達促進KLE細胞的增殖(P=0.025,MTT; P=0.011,克隆形成)、遷移(P=0.026)、侵襲(P=0.019)能力；下調EMX2后更多的KLE細胞進入S期；下調EMX2表達對KLE細胞的凋亡無明顯影響。 (3)裸鼠成瘤實驗中，EMX2過表達可明顯抑制RL95-2移植瘤的生長速度(P=0.005)和腫瘤重量(P=0.023)。結論：體內外實驗證明EMX2可抑制子宮內膜癌的增殖、遷移、侵襲等惡性行為。 第四部分Wnt信號通路介導EMX2基因的抑癌作用 目的：探討EMX2基因通過Wnt信號通路發(fā)揮其抑癌作用的分子機制。 方法：用pcDNA3.1-EMX2上調RL95-2和siRNA-EMX2下調KLE細胞中EMX2表達后，用realtime PCR和western blot檢測細胞內Wnt信號分子β-catenin及其靶基因cyclin D1的表達改變。分別用Wnt信號通路抑制劑XAV-939和激動劑LiCl預處理子宮內膜癌細胞后，再次用pcDNA3.1-EMX2上調RL95-2和siRNA-EMX2下調KLE細胞中EMX2表達，觀察EMX2表達改變對細胞增殖、周期的影響。 結果： (1)上調EMX2可抑制RL95-2細胞中β-catenin及其下游因子cyclin D1的表達；反之，下調EMX2后KLE細胞中β-catenin、cyclin D1表達上升。 (2) EMX2過表達對β-catenin/cyclin D1的抑制作用可被LiCl拮抗,同時EMX2對RL95-2細胞的增殖、周期抑制被解除；EMX2失活對β-catenin/cyclin D1的激活作用可被XAV-939明顯削弱,同時EMX2失活對KLE細胞的促增殖、周期作用被XAV-939消除。 結論：Wnt信號作為關鍵通路介導了EMX2基因在子宮內膜癌中發(fā)揮抑癌作用。
[Abstract]:In China, endometrial carcinoma is one of the most common malignant tumors in the female genital tract, and its incidence is increasing year by year. About 25% of cases of endometrial carcinoma are advanced at the time of diagnosis. The objective response rate to chemotherapy and endocrine therapy is very low and the prognosis is poor. The submechanism is not yet clear.
It is reported that the homeobox transcription gene EMX2 plays a key role in the development of female reproductive system. The reproductive tract (uterus, cervix, vagina, etc.) of female mice is completely absent. EMX2 can antagonize the hyperplasia of normal endometrial epithelial cells in vitro. Preliminary studies showed that EMX2 expression in about 30% (10/30) of endometrial carcinoma tissues was significantly decreased, suggesting that the inactivation of EMX2 gene may be involved in the occurrence and development of endometrial carcinoma.
Based on the above findings, the biological function and mechanism of EMX2 gene in endometrial carcinoma were discussed from the following four aspects: (1) To detect the expression of EMX2 mRNA and protein in tissues and cell lines, and to analyze the correlation between EMX2 mRNA and the clinicopathological parameters of patients; (2) Methylation specific PCR (MSP) and DNA assay. The frequency of hypermethylation in the promoter region of EMX2 gene in endometrial carcinoma was detected by sequencing and other techniques to determine whether DNA methylation could result in the decrease of EMX2 expression and to verify it in the corresponding endometrial carcinoma cells. (3) The expression of EMX2 was down-regulated in endometrial carcinoma cells, and the expression of EMX2 was cloned by MTT, wound healing, Transwell, and so on. Flow cytometry, real-time PCR and Western blot were used to detect the effects of EMX2 gene on proliferation, migration and invasion of endometrial cancer cells; a nude mouse model of endometrial cancer was established to verify the inhibitory effect of EMX2 on endometrial cancer in vivo (; 4) real-time PCR and Western blot were used to detect the effects of EMX2 on Wnt signaling pathway. Wnt signaling pathway inhibitors (XAV-939) and agonists (LiCl) validate whether Wnt signaling is a key downstream pathway for the antitumor effect of EMX2 gene.
Objective: To detect the expression of EMX2 in human endometrial carcinoma tissue and immortalized endometrial carcinoma cell line, and to explore the correlation between the expression of EMX2 and clinicopathological parameters.
Methods: Immunohistochemical technique was used to detect the expression and localization of EMX2 protein in normal endometrial tissues (n=25) and endometrial carcinoma tissues (n=122). Correlation.
Result:
(1) Compared with normal endometrium, the expression of EMX2 protein in 48.4% (59/122) of endometrial carcinoma was decreased (P 0.001). The expression of EMX2 was closely related to tumor stage (P = 0.023), grade (P = 0.016) and deep myometrial invasion (P = 0.04), but was not related to age, lymphatic vascular space invasion, lymph node invasion and estrogen and progesterone receptor status.
(2) In normal endometrial tissues, EMX2 protein was mainly localized in the nucleus and partly accompanied by cytoplasmic staining.
(3) The expression of EMX2 was high in KLE and Ishikawa cells, but very low in RL95-2, AN3CA and SPEC-2 cells. Conclusion: EMX2 inactivation was frequently found in endometrial carcinoma tissues and was significantly associated with the malignancy of the tumor, suggesting that EMX2 inactivation may be involved in the occurrence and progression of endometrial carcinoma.
The second part is the mechanism of EMX2 gene inactivation in endometrial carcinoma.
Objective: To investigate the mechanism of EMX2 gene inactivation in human endometrial carcinoma.
METHODS: Methylation-specific PCR (MSP) and sequencing were used to detect the frequency of methylation in the promoter region of EMX2 gene in endometrial carcinoma cell line (n=4), normal endometrium (n=22) and endometrial carcinoma tissue (n=79); 5-AZA-dC was used to treat endometrial carcinoma cells, real-time PCR and Western blot were used to detect the frequency of methylation. Changes of EMX2mRNA and protein levels in cells.
Result:
(1) The incidence of hypermethylation of EMX2 promoter was 4.5% (1/22) and 39.2% (32/79, P 0.001) in normal endometrial tissues and endometrial carcinoma tissues respectively, and hypermethylation was significantly associated with the inactivation of EMX2 expression (P 0.001).
(2) EMX2 promoter hypermethylation was found only in RL95-2 cells, and the expression of EMX2 mRNA and protein in RL95-2 cells increased significantly after 12 hours of treatment with 1 mu M5-AZA-dC, while the expression of EMX2 in the other three cells remained unchanged.
Conclusion: Promoter hypermethylation is an important factor leading to the inactivation of EMX2 gene in endometrial carcinoma, and EMX2 may be a tumor suppressor gene.
Objective: To investigate the effects of EMX2 on proliferation, apoptosis, cycle, migration, invasion and tumorigenesis of endometrial carcinoma cells. Methods: RL95-2 and KLE cells were transfected by pcDNA3.1-EMX2, siRNA-EMX2 and their negative control, respectively. Formation assay was used to detect cell proliferation; flow cytometry was used to detect cell apoptosis and cell cycle changes; scratch assay was used to detect cell migration; Transwell chamber assay was used to detect cell invasiveness. The effect of EMX2 on tumor formation was verified in nude mice.
Result:
(1) Detection of transfection effect: After transfection of pcDNA3.1-EMX2 plasmid, the levels of EMX2 mRNA and protein in RL95-2 cells increased significantly; after transfection of siRNA-EMX2, the levels of EMX2 mRNA and protein in KLE cells decreased by more than 75%.
(2) Upregulation of EMX2 expression significantly inhibited the proliferation (P = 0.002, MTT; P = 0.03, cloning), migration (P = 0.018) and invasion (P = 0.039) of RL95-2 cells; EMX2 could block RL95-2 cells in G1 phase, but EMX2 did not increase the apoptosis rate of RL95-2 cells. Downregulation of EMX2 expression promoted the proliferation of KLE cells (P = 0.025, MTT; P = 0.011, cloning). Formation, migration (P = 0.026), invasion (P = 0.019); more KLE cells entered S phase after down-regulation of EMX2; down-regulation of EMX2 expression had no significant effect on KLE cell apoptosis.
(3) Overexpression of EMX2 significantly inhibited the growth rate (P = 0.005) and tumor weight (P = 0.023) of RL95-2 transplanted tumors in nude mice.
The fourth part of the Wnt signaling pathway mediates the suppressive effect of EMX2 gene.
Objective: To explore the molecular mechanism of EMX2 gene suppressing cancer through Wnt signaling pathway.
Methods: EMX2 expression in KLE cells was up-regulated by pcDNA3.1-EMX2 and down-regulated by siRNA-EMX2. The expression of Wnt signaling molecule beta-catenin and its target gene cyclin D 1 were detected by realtime PCR and Western blot. After pretreatment with Wnt signaling pathway inhibitor XAV-939 and activator LiCl respectively, endometrial carcinoma cells were treated with pcDNA3.1 again. EMX2 up-regulated RL95-2 and siRNA-EMX2 down-regulated EMX2 expression in KLE cells, and observed the effect of EMX2 expression on cell proliferation and cell cycle.
Result:
(1) Upregulation of EMX2 inhibited the expression of beta-catenin and its downstream factor cyclin D1 in RL95-2 cells, whereas down-regulation of EMX2 increased the expression of beta-catenin and cyclin D1 in KLE cells.
(2) The inhibitory effect of EMX2 overexpression on beta-catenin/cyclin D1 was antagonized by LiCl, and the proliferation of RL95-2 cells was relieved by EMX2. The activation of EMX2 inactivation on beta-catenin/cyclin D1 was significantly weakened by XAV-939, and the proliferation of KLE cells was promoted by EMX2 inactivation, and the cycle effect was eliminated by XAV-939.
Conclusion: Wnt signaling as a key pathway mediates the role of EMX2 gene in endometrial carcinoma.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.33

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