子宫内膜癌中EMX2-Wnt通路的生物学功能及其机制研究
发布时间：2018-08-13 12:39
【摘要】：在我国，子宫内膜癌是女性生殖道最常见的恶性肿瘤之一，且其发病率逐年持续上升。约25%的子宫内膜癌病例在确诊时已属晚期，对化疗和内分泌治疗的客观反应率很低，预后不佳。然而,迄今我们对子宫内膜癌发生、进展及侵袭转移的分子机制尚不清楚。 据报道,同源盒转录基因EMX2在雌性生殖系统发育中起关键作用，Emx2-/-雌性小鼠的生殖道（子宫、宫颈、阴道等）完全缺如。在绝经前子宫内膜中，，EMX2的蛋白表达量随月经周期发生规律性改变，而绝经后子宫内膜中EMX2呈持续性高表达；同时，体外实验证实EMX2可拮抗正常内膜上皮细胞的过度增殖。课题组预实验发现约30%（10/30）的子宫内膜癌组织中EMX2表达显著下降，提示EMX2基因失活可能参与了子宫内膜癌的发生发展等过程。 基于以上发现，本课题拟从以下四个方面对子宫内膜癌中EMX2基因的生物学功能及其作用机制进行探讨：（1）检测组织和细胞系EMX2mRNA和蛋白的表达水平,分析其与患者各临床病理参数之间的相关性；（2）应用甲基化特异性PCR(MSP)及DNA测序等技术检测子宫内膜癌中EMX2基因启动子区高甲基化的发生频率，确定DNA甲基化是否可导致EMX2表达下降，并在相应的子宫内膜癌细胞中进行验证；（3）在子宫内膜癌细胞中分别上、下调EMX2的表达，采用MTT、克隆形成、wound healing、Transwell、流式细胞术、real time PCR和western blot等方法检测EMX2基因对细胞增殖、迁移、侵袭等生物学行为的影响；建立子宫内膜癌裸鼠荷瘤模型，体内验证EMX2对子宫内膜癌的抑制作用（；4）采用real time PCR、western blot检测EMX2对Wnt信号通路的影响，应用Wnt信号通路抑制剂(XAV-939)和激动剂(LiCl)验证Wnt信号是否为EMX2基因抗肿瘤作用的关键下游通路。 第一部分EMX2在子宫内膜癌组织和细胞系中的表达目的：检测人子宫内膜癌组织和永生化子宫内膜癌细胞系中EMX2的表达水平，探讨EMX2表达与患者临床病理参数之间的相关性。 方法：应用免疫组织化学方法技术检测正常内膜组织（n=25）、子宫内膜癌组织（n=122）中EMX2蛋白的表达和定位。应用real time PCR、western blot检测子宫内膜癌细胞系中EMX2mRNA和蛋白的表达水平。应用SPSS软件分析EMX2表达与患者临床病理参数之间的相关性。 结果： (1)与正常内膜相比，48.4%（59/122）的子宫内膜癌组织中EMX2蛋白表达下降（P 0.001）。EMX2表达下降与肿瘤分期(P=0.023)、分级(P=0.016)、深肌层浸润(P=0.04)密切相关，与年龄、淋巴脉管间隙浸润、淋巴结侵犯以及雌孕激素受体状态无相关性。 (2)在正常内膜组织中，EMX2蛋白主要定位于细胞核，部分可伴细胞浆同时着色。在子宫内膜癌组织中未见EMX2蛋白着色部位的异常。 (3) KLE、Ishikawa细胞中EMX2表达较高，RL95-2、AN3CA、SPEC-2细胞中EMX2表达极低。结论：EMX2在子宫内膜癌组织中频发失活，且与肿瘤恶性程度明显相关，提示EMX2失活可能参与了子宫内膜癌的发生、进展。 第二部分子宫内膜癌中EMX2基因失活机制 目的：探讨人子宫内膜癌中EMX2基因失活的机制。 方法：应用甲基化特异性PCR(MSP)及测序技术检测子宫内膜癌细胞系（n=4）、正常内膜（n=22）和子宫内膜癌组织（n=79）中EMX2基因启动子区发生甲基化的频率；应用甲基化逆转药物5-AZA-dC处理子宫内膜癌细胞，采用real time PCR、western blot分别检测细胞内EMX2mRNA和蛋白水平的改变。 结果： (1)在正常内膜和子宫内膜癌组织中，EMX2基因启动子高甲基化的发生率分别为4.5%（1/22）和39.2%（32/79，P 0.001），且高甲基化与EMX2表达失活明显相关(P 0.001)。 (2)仅RL95-2细胞中存在EMX2启动子高甲基化；经1μM5-AZA-dC处理12小时后，RL95-2细胞中EMX2mRNA和蛋白表达均明显上升，而其他3种细胞中EMX2表达无明显变化。 结论：启动子高甲基化是导致子宫内膜癌中EMX2基因失活的一个重要因素，同时也提示EMX2可能是一个抑癌基因。 第三部分EMX2基因对子宫内膜癌细胞生物学行为的影响目的：探讨EMX2对子宫内膜癌细胞增殖、凋亡、周期、迁移、侵袭和成瘤能力的影响。方法：使用pcDNA3.1-EMX2、siRNA-EMX2及它们的阴性对照对RL95-2和KLE细胞分别进行瞬时转染，应用MTT、克隆形成实验检测细胞增殖能力；应用流式细胞术检测细胞凋亡、周期的改变；应用划痕实验检测细胞迁移能力；应用Transwell小室实验检测细胞侵袭能力。应用pcDNA3.1-EMX2转染RL95-2细胞并用G418筛选、构建EMX2稳定高表达细胞株，应用该细胞株在裸鼠体内验证EMX2对肿瘤形成的影响。 结果： (1)转染效果检测：瞬时转染pcDNA3.1-EMX2质粒后，RL95-2细胞中EMX2mRNA和蛋白水平均显著升高；瞬时转染siRNA-EMX2后，KLE细胞中EMX2mRNA和蛋白水平下降超过75%。 (2)上调EMX2表达后，RL95-2细胞的增殖(P=0.002, MTT; P=0.03,克隆形成)、迁移(P=0.018)、侵袭(P=0.039)能力受到明显抑制；同时EMX2可将RL95-2细胞阻滞在G1期；但EMX2并不增加RL95-2细胞的凋亡率。而下调EMX2表达促进KLE细胞的增殖(P=0.025,MTT; P=0.011,克隆形成)、迁移(P=0.026)、侵袭(P=0.019)能力；下调EMX2后更多的KLE细胞进入S期；下调EMX2表达对KLE细胞的凋亡无明显影响。 (3)裸鼠成瘤实验中，EMX2过表达可明显抑制RL95-2移植瘤的生长速度(P=0.005)和肿瘤重量(P=0.023)。结论：体内外实验证明EMX2可抑制子宫内膜癌的增殖、迁移、侵袭等恶性行为。 第四部分Wnt信号通路介导EMX2基因的抑癌作用 目的：探讨EMX2基因通过Wnt信号通路发挥其抑癌作用的分子机制。 方法：用pcDNA3.1-EMX2上调RL95-2和siRNA-EMX2下调KLE细胞中EMX2表达后，用realtime PCR和western blot检测细胞内Wnt信号分子β-catenin及其靶基因cyclin D1的表达改变。分别用Wnt信号通路抑制剂XAV-939和激动剂LiCl预处理子宫内膜癌细胞后，再次用pcDNA3.1-EMX2上调RL95-2和siRNA-EMX2下调KLE细胞中EMX2表达，观察EMX2表达改变对细胞增殖、周期的影响。 结果： (1)上调EMX2可抑制RL95-2细胞中β-catenin及其下游因子cyclin D1的表达；反之，下调EMX2后KLE细胞中β-catenin、cyclin D1表达上升。 (2) EMX2过表达对β-catenin/cyclin D1的抑制作用可被LiCl拮抗,同时EMX2对RL95-2细胞的增殖、周期抑制被解除；EMX2失活对β-catenin/cyclin D1的激活作用可被XAV-939明显削弱,同时EMX2失活对KLE细胞的促增殖、周期作用被XAV-939消除。 结论：Wnt信号作为关键通路介导了EMX2基因在子宫内膜癌中发挥抑癌作用。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
【参考文献】
相关期刊论文 前3条
1 王永学;潘凌亚;黄惠芳;沈铿;郎景和;;年轻子宫内膜癌患者孕激素保守治疗临床分析[J];中华肿瘤防治杂志;2011年07期
2 易晓芳;郑文新;;子宫内膜癌的分型及其临床意义[J];中国实用妇科与产科杂志;2008年01期
3 徐望红 ,项永兵 ,金凡 ,周淑贞 ,方茹蓉 ,阮志贤 ,孙璐 ,高玉堂;上海市区女性生殖系统恶性肿瘤发病趋势分析[J];肿瘤;2003年04期
本文编号：2181022
本文链接：https://www.wllwen.com/yixuelunwen/fuchankeerkelunwen/2181022.html
最近更新
教材专著
