子宮內(nèi)膜癌中EMX2-Wnt通路的生物學(xué)功能及其機(jī)制研究
發(fā)布時(shí)間：2018-08-13 12:39
【摘要】：在我國(guó)，子宮內(nèi)膜癌是女性生殖道最常見(jiàn)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(nèi)膜癌病例在確診時(shí)已屬晚期，對(duì)化療和內(nèi)分泌治療的客觀(guān)反應(yīng)率很低，預(yù)后不佳。然而,迄今我們對(duì)子宮內(nèi)膜癌發(fā)生、進(jìn)展及侵襲轉(zhuǎn)移的分子機(jī)制尚不清楚。 據(jù)報(bào)道,同源盒轉(zhuǎn)錄基因EMX2在雌性生殖系統(tǒng)發(fā)育中起關(guān)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(nèi)膜中，，EMX2的蛋白表達(dá)量隨月經(jīng)周期發(fā)生規(guī)律性改變，而絕經(jīng)后子宮內(nèi)膜中EMX2呈持續(xù)性高表達(dá)；同時(shí)，體外實(shí)驗(yàn)證實(shí)EMX2可拮抗正常內(nèi)膜上皮細(xì)胞的過(guò)度增殖。課題組預(yù)實(shí)驗(yàn)發(fā)現(xiàn)約30%（10/30）的子宮內(nèi)膜癌組織中EMX2表達(dá)顯著下降，提示EMX2基因失活可能參與了子宮內(nèi)膜癌的發(fā)生發(fā)展等過(guò)程。 基于以上發(fā)現(xiàn)，本課題擬從以下四個(gè)方面對(duì)子宮內(nèi)膜癌中EMX2基因的生物學(xué)功能及其作用機(jī)制進(jìn)行探討：（1）檢測(cè)組織和細(xì)胞系EMX2mRNA和蛋白的表達(dá)水平,分析其與患者各臨床病理參數(shù)之間的相關(guān)性；（2）應(yīng)用甲基化特異性PCR(MSP)及DNA測(cè)序等技術(shù)檢測(cè)子宮內(nèi)膜癌中EMX2基因啟動(dòng)子區(qū)高甲基化的發(fā)生頻率，確定DNA甲基化是否可導(dǎo)致EMX2表達(dá)下降，并在相應(yīng)的子宮內(nèi)膜癌細(xì)胞中進(jìn)行驗(yàn)證；（3）在子宮內(nèi)膜癌細(xì)胞中分別上、下調(diào)EMX2的表達(dá)，采用MTT、克隆形成、wound healing、Transwell、流式細(xì)胞術(shù)、real time PCR和western blot等方法檢測(cè)EMX2基因?qū)?xì)胞增殖、遷移、侵襲等生物學(xué)行為的影響；建立子宮內(nèi)膜癌裸鼠荷瘤模型，體內(nèi)驗(yàn)證EMX2對(duì)子宮內(nèi)膜癌的抑制作用（；4）采用real time PCR、western blot檢測(cè)EMX2對(duì)Wnt信號(hào)通路的影響，應(yīng)用Wnt信號(hào)通路抑制劑(XAV-939)和激動(dòng)劑(LiCl)驗(yàn)證Wnt信號(hào)是否為EMX2基因抗腫瘤作用的關(guān)鍵下游通路。 第一部分EMX2在子宮內(nèi)膜癌組織和細(xì)胞系中的表達(dá)目的：檢測(cè)人子宮內(nèi)膜癌組織和永生化子宮內(nèi)膜癌細(xì)胞系中EMX2的表達(dá)水平，探討EMX2表達(dá)與患者臨床病理參數(shù)之間的相關(guān)性。 方法：應(yīng)用免疫組織化學(xué)方法技術(shù)檢測(cè)正常內(nèi)膜組織（n=25）、子宮內(nèi)膜癌組織（n=122）中EMX2蛋白的表達(dá)和定位。應(yīng)用real time PCR、western blot檢測(cè)子宮內(nèi)膜癌細(xì)胞系中EMX2mRNA和蛋白的表達(dá)水平。應(yīng)用SPSS軟件分析EMX2表達(dá)與患者臨床病理參數(shù)之間的相關(guān)性。 結(jié)果： (1)與正常內(nèi)膜相比，48.4%（59/122）的子宮內(nèi)膜癌組織中EMX2蛋白表達(dá)下降（P 0.001）。EMX2表達(dá)下降與腫瘤分期(P=0.023)、分級(jí)(P=0.016)、深肌層浸潤(rùn)(P=0.04)密切相關(guān)，與年齡、淋巴脈管間隙浸潤(rùn)、淋巴結(jié)侵犯以及雌孕激素受體狀態(tài)無(wú)相關(guān)性。 (2)在正常內(nèi)膜組織中，EMX2蛋白主要定位于細(xì)胞核，部分可伴細(xì)胞漿同時(shí)著色。在子宮內(nèi)膜癌組織中未見(jiàn)EMX2蛋白著色部位的異常。 (3) KLE、Ishikawa細(xì)胞中EMX2表達(dá)較高，RL95-2、AN3CA、SPEC-2細(xì)胞中EMX2表達(dá)極低。結(jié)論：EMX2在子宮內(nèi)膜癌組織中頻發(fā)失活，且與腫瘤惡性程度明顯相關(guān)，提示EMX2失活可能參與了子宮內(nèi)膜癌的發(fā)生、進(jìn)展。 第二部分子宮內(nèi)膜癌中EMX2基因失活機(jī)制 目的：探討人子宮內(nèi)膜癌中EMX2基因失活的機(jī)制。 方法：應(yīng)用甲基化特異性PCR(MSP)及測(cè)序技術(shù)檢測(cè)子宮內(nèi)膜癌細(xì)胞系（n=4）、正常內(nèi)膜（n=22）和子宮內(nèi)膜癌組織（n=79）中EMX2基因啟動(dòng)子區(qū)發(fā)生甲基化的頻率；應(yīng)用甲基化逆轉(zhuǎn)藥物5-AZA-dC處理子宮內(nèi)膜癌細(xì)胞，采用real time PCR、western blot分別檢測(cè)細(xì)胞內(nèi)EMX2mRNA和蛋白水平的改變。 結(jié)果： (1)在正常內(nèi)膜和子宮內(nèi)膜癌組織中，EMX2基因啟動(dòng)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(dá)失活明顯相關(guān)(P 0.001)。 (2)僅RL95-2細(xì)胞中存在EMX2啟動(dòng)子高甲基化；經(jīng)1μM5-AZA-dC處理12小時(shí)后，RL95-2細(xì)胞中EMX2mRNA和蛋白表達(dá)均明顯上升，而其他3種細(xì)胞中EMX2表達(dá)無(wú)明顯變化。 結(jié)論：?jiǎn)?dòng)子高甲基化是導(dǎo)致子宮內(nèi)膜癌中EMX2基因失活的一個(gè)重要因素，同時(shí)也提示EMX2可能是一個(gè)抑癌基因。 第三部分EMX2基因?qū)ψ訉m內(nèi)膜癌細(xì)胞生物學(xué)行為的影響目的：探討EMX2對(duì)子宮內(nèi)膜癌細(xì)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(duì)照對(duì)RL95-2和KLE細(xì)胞分別進(jìn)行瞬時(shí)轉(zhuǎn)染，應(yīng)用MTT、克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力；應(yīng)用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡、周期的改變；應(yīng)用劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力；應(yīng)用Transwell小室實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力。應(yīng)用pcDNA3.1-EMX2轉(zhuǎn)染RL95-2細(xì)胞并用G418篩選、構(gòu)建EMX2穩(wěn)定高表達(dá)細(xì)胞株，應(yīng)用該細(xì)胞株在裸鼠體內(nèi)驗(yàn)證EMX2對(duì)腫瘤形成的影響。 結(jié)果： (1)轉(zhuǎn)染效果檢測(cè)：瞬時(shí)轉(zhuǎn)染pcDNA3.1-EMX2質(zhì)粒后，RL95-2細(xì)胞中EMX2mRNA和蛋白水平均顯著升高；瞬時(shí)轉(zhuǎn)染siRNA-EMX2后，KLE細(xì)胞中EMX2mRNA和蛋白水平下降超過(guò)75%。 (2)上調(diào)EMX2表達(dá)后，RL95-2細(xì)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(shí)EMX2可將RL95-2細(xì)胞阻滯在G1期；但EMX2并不增加RL95-2細(xì)胞的凋亡率。而下調(diào)EMX2表達(dá)促進(jìn)KLE細(xì)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(diào)EMX2后更多的KLE細(xì)胞進(jìn)入S期；下調(diào)EMX2表達(dá)對(duì)KLE細(xì)胞的凋亡無(wú)明顯影響。 (3)裸鼠成瘤實(shí)驗(yàn)中，EMX2過(guò)表達(dá)可明顯抑制RL95-2移植瘤的生長(zhǎng)速度(P=0.005)和腫瘤重量(P=0.023)。結(jié)論：體內(nèi)外實(shí)驗(yàn)證明EMX2可抑制子宮內(nèi)膜癌的增殖、遷移、侵襲等惡性行為。 第四部分Wnt信號(hào)通路介導(dǎo)EMX2基因的抑癌作用 目的：探討EMX2基因通過(guò)Wnt信號(hào)通路發(fā)揮其抑癌作用的分子機(jī)制。 方法：用pcDNA3.1-EMX2上調(diào)RL95-2和siRNA-EMX2下調(diào)KLE細(xì)胞中EMX2表達(dá)后，用realtime PCR和western blot檢測(cè)細(xì)胞內(nèi)Wnt信號(hào)分子β-catenin及其靶基因cyclin D1的表達(dá)改變。分別用Wnt信號(hào)通路抑制劑XAV-939和激動(dòng)劑LiCl預(yù)處理子宮內(nèi)膜癌細(xì)胞后，再次用pcDNA3.1-EMX2上調(diào)RL95-2和siRNA-EMX2下調(diào)KLE細(xì)胞中EMX2表達(dá)，觀(guān)察EMX2表達(dá)改變對(duì)細(xì)胞增殖、周期的影響。 結(jié)果： (1)上調(diào)EMX2可抑制RL95-2細(xì)胞中β-catenin及其下游因子cyclin D1的表達(dá)；反之，下調(diào)EMX2后KLE細(xì)胞中β-catenin、cyclin D1表達(dá)上升。 (2) EMX2過(guò)表達(dá)對(duì)β-catenin/cyclin D1的抑制作用可被LiCl拮抗,同時(shí)EMX2對(duì)RL95-2細(xì)胞的增殖、周期抑制被解除；EMX2失活對(duì)β-catenin/cyclin D1的激活作用可被XAV-939明顯削弱,同時(shí)EMX2失活對(duì)KLE細(xì)胞的促增殖、周期作用被XAV-939消除。 結(jié)論：Wnt信號(hào)作為關(guān)鍵通路介導(dǎo)了EMX2基因在子宮內(nèi)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33
本文編號(hào)：2181022
[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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33
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