SOX17和CyclinD1在子宫内膜样腺癌中的表达及5-AZA对其表达的影响
发布时间：2018-07-20 20:58
【摘要】：背景与目的子宫内膜癌是妇科常见的恶性肿瘤,近年来其发病率特别是年轻患者的发病率呈上升趋势。子宫内膜癌的发病机制尚不明确,表观遗传学的改变在肿瘤的发生发展过程中起重要作用。就目前而言,SOX17在子宫内膜癌发生发展中的作用尚不明确,本研究旨在探讨SOX17与子宫内膜癌发生发展的关系。SOX17是经典Wnt信号通路的重要拮抗剂,通过抑制β-catenin/TCF依赖性转录及癌细胞增殖和集落的形成,从而调节Wnt/β-catenin信号转导。多项研究表明,SOX17启动子的高甲基化是SOX17基因失活的主要原因,其在组织中呈现低表达率,进而导致癌细胞的增殖。另有研究表明SOX17的抑制肿瘤机制可能与其使细胞停滞在G1/S期,诱导凋亡、调节肿瘤细胞的血管生成的作用有关。由此得出SOX17可能在人类抑癌作用中发挥作用,但其在人类子宫内膜癌中的潜在作用尚未阐明。人体肿瘤是一种细胞周期性疾病,细胞周期素D1(Cyclin D1)作为细胞周期的正向调节因子,促进了肿瘤细胞的增殖。甲基转移酶抑制剂5-氮杂胞苷(5-AZA)能够通过去甲基化作用使过甲基化的基因重新表达,从而恢复其抑癌功能。我们的前期研究已表明SOX17基因在子宫内膜癌组织及细胞株中表达下降,与其启动子处于高甲基化状态有关,本研究利用去甲基化药物5-AZA作用子宫内膜样腺癌细胞,实时荧光定量PCR检测药物作用前后SOX17和Cyclin D1基因的表达,同时对子宫内膜样腺癌组织中SOX17和Cyclin D1表达水平进行检测,旨在探讨SOX17在子宫内膜样腺癌的发生发展中的作用及其可能机制。材料与方法1.实验材料1.1细胞来源:人子宫内膜样腺癌细胞株HEC1A中分化,由郑州大学第一附属医院重点实验室馈赠。1.2组织标本来源与病例资料:收集2014年7月~2016年12月于郑州大学第二附属医院行子宫切除的子宫内膜组织标本,术中标本离体后立即取材,迅速置于-80℃保存。所取标本包括30例子宫内膜样腺癌组织和10例正常增生期子宫内膜组织,所有标本均经郑州大学第二附属医院病理科确诊。2.实验方法2.1实验分组:所收集标本共分两组,子宫内膜样腺癌组30例,正常增生期子宫内膜组10例(因宫颈上皮内瘤变手术切除子宫的子宫内膜组织),入组患者术前均未进行放疗、化疗、激素免疫治疗及其他抗肿瘤治疗,且排除合并其他部位肿瘤的患者,组织标本均由病理科医师镜下观察确诊。2.2正常增生期子宫内膜组织和子宫内膜样腺癌组织中SOX17、β-catenin和Cyclin D1基因的m RNA表达情况:采用实时荧光定量PCR技术对上述30例子宫内膜样腺癌组织和10例正常增生期子宫内膜组织中SOX17、β-catenin和Cyclin D1基因表达情况进行检测。2.3 5-AZA对子宫内膜样腺癌细胞增殖的影响:采用细胞培养技术,用不同浓度5-AZA处理子宫内膜样腺癌细胞株HEC1A,检测细胞生长抑制情况。使用MTT法检测24h、48h、72h不同浓度的5-AZA对HEC1A细胞的生长抑制作用的影响;采用实时荧光定量PCR检测处理前后HEC1A中三种基因的m RNA表达情况。3.统计方法应用SPSS 23.0软件包对数据进行统计学分析,定量资料描述应用(?),组间采用独立样本t检验,单因素方差分析及Pearson相关分析,以P0.05差异有统计学意义,以α=0.05为检验水准。结果1正常子宫内膜组织和子宫内膜样腺癌组织中SOX17、β-catenin和Cyclin D1基因的m RNA表达情况。本实验检测结果显示,SOX17基因在子宫内膜样腺癌组织中表达水平下降,同时β-catenin和Cyclin D1基因在子宫内膜样腺癌组织中表达升高,与正常增生期子宫内膜组织相比,差异均有统计学意义(P0.05)。在30例子宫内膜样腺癌组织标本中,SOX17、β-catenin和Cyclin D1基因m RNA相对表达量在组织学分级、淋巴结转移及FIGO分期中表达差异均有统计学意义(P0.05)。其中β-catenin及Cyclin D1的m RNA相对表达量在肌层浸润深度中差异有统计学意义(P0.05)。2 5-AZA对子宫内膜样腺癌细胞增殖的影响使用不同浓度5-AZA处理HEC1A细胞72h后,经MTT法检测,细胞的生长抑制作用差异具有统计学意义(P0.05),且处理72h后对细胞生长作用抑制最明显(IC50=12.033)。随着处理时间的增长,不同浓度5-AZA对HEC1A细胞生长抑制效果增强,且差异有统计学意义(P0.05);5-AZA处理子宫内膜样腺癌细胞后SOX17基因表达较处理前明显增高(P0.05),同时β-catenin和Cyclin D1基因的m RNA表达明显降低(P0.05)。3子宫内膜样腺癌组织中SOX17和Cyclin D1基因的相关性分析本文对30例子宫内膜样腺癌组织中Cyclin D1、β-catenin及SOX17基因的m RNA相对表达量进行pearson两两相关性分析发现,SOX17及Cyclin D1在子宫内膜样腺癌组织中的表达呈弱负相关(r=-0.353,P0.05),SOX17与β-catenin在子宫内膜样腺癌组织中表达呈负相关(r=-0.463,P0.05),β-catenin和Cyclin D1的表达在子宫内膜样腺癌组织中呈明显正相关(r=0.863,P0.001)。结论1.SOX17基因可能作为一种潜在的肿瘤抑制因子,通过下调Cyclin D1及β-catenin基因的表达来实现其肿瘤抑制作用,为子宫内膜样腺癌的基因治疗提供新的思路。2.去甲基化药物5-AZA可提高SOX17基因在子宫内膜样腺癌细胞株中的表达,并明显抑制子宫内膜癌细胞的增殖,在未来子宫内膜癌的临床治疗中,其可能成为潜在有效的治疗药物,通过作用SOX17基因来实现其抗癌作用。
[Abstract]:Background and objective endometrial carcinoma is a common malignant tumor in gynecology. In recent years, the incidence of the incidence of endometriosis, especially in young patients, is on the rise. The pathogenesis of endometrial cancer is not clear, and epigenetic changes play an important role in the development of tumor. At present, SOX17 is developing in endometrial cancer. The role of SOX17 is not yet clear. The aim of this study is to explore the relationship between SOX17 and the development of endometrial carcinoma..SOX17 is an important antagonist of the classic Wnt signaling pathway, which regulates the signal transduction of Wnt/ beta -catenin by inhibiting beta -catenin/TCF dependent transcription and cancer cell proliferation and colony formation. Several studies have shown that the high level of SOX17 promoter is high. It is the main reason for the inactivation of SOX17 gene, which shows low rate of expression in the tissue and leads to the proliferation of cancer cells. Other studies have shown that the inhibition of tumor mechanism by SOX17 may be related to the effect of cell stagnation at G1/S stage, inducing apoptosis and regulating the angiogenesis of tumor cells. Thus, SOX17 may be in human tumor suppressor effect. It plays a role, but its potential role in human endometrial cancer has not been elucidated. Human tumor is a cyclical disease, cyclin D1 (Cyclin D1), as a positive regulator of cell cycle, promotes the proliferation of tumor cells. The methyltransferase inhibitor 5- nitrogen heterocytidine (5-AZA) can be induced by demethylation. Our previous study has shown that the expression of SOX17 gene in endometrial carcinoma tissues and cell lines is decreased, and that the promoter is in the state of hypermethylation. This study uses demethylation drug 5-AZA to act on endometrioid adenocarcinoma cells, real-time fluorescence quantitative PCR detection The expression of SOX17 and Cyclin D1 genes before and after drug action, and the expression of SOX17 and Cyclin D1 in endometrioid adenocarcinoma tissue, to explore the role of SOX17 in the development of endometrioid adenocarcinoma and its possible mechanism. Materials and methods 1. experimental material 1.1 cell sources: human endometrioid adenocarcinoma cell line HE C1A differentiation, from the First Affiliated Hospital of the Zhengzhou University, the Key Laboratory of the First Affiliated Hospital of the Zhengzhou University,.1.2 tissue specimen source and case data: collection of endometrium tissue specimens from the Second Affiliated Hospital of Zhengzhou University, July 2014, at the Second Affiliated Hospital of Zhengzhou University. The specimens were harvested immediately after the operation in vitro, and were quickly stored at -80 C. The samples included 30 cases of uterus. Endometrioid adenocarcinoma and 10 cases of normal proliferative endometrium, all specimens were divided into two groups, 30 cases of endometrioid adenocarcinoma, 10 cases of endometrioid endometrium endometrium, and 10 cases of endometrioid endometrium in normal hyperplastic period of endometrium. All specimens were divided into 2.1 groups, which were confirmed by the pathology department of the Second Affiliated Hospital of Zhengzhou University. The specimens were divided into two groups, 30 cases of endometrioid adenocarcinoma, and 10 cases of normal hyperplasia. No radiotherapy, chemotherapy, hormone immunotherapy, and other antitumor treatment were performed before operation, and patients with other tumors were excluded. The tissue specimens were examined by a pathologist to confirm the diagnosis of SOX17, beta -catenin and Cyclin D1 in endometrioid adenocarcinoma tissue of.2.2 and endometrioid adenocarcinoma. The expression of M RNA: the effects of SOX17, beta -catenin and Cyclin D1 on the proliferation of endometrioid adenocarcinoma cells in 30 cases of endometrioid adenocarcinoma and 10 normal endometrium tissues of the above 30 cases of endometrioid adenocarcinoma and 10 cases of normal endometrium were detected by real-time fluorescent quantitative PCR: cell culture and 5-A with different concentrations of 5-A. ZA treatment of endometrioid adenocarcinoma cell line HEC1A was used to detect cell growth inhibition. The effects of 5-AZA on the growth inhibition of HEC1A cells with different concentrations of 24h, 48h, 72h were detected by MTT method. The m RNA table of three genes in HEC1A before and after the real-time fluorescence quantitative PCR detection was used for the logarithmic logarithm of the 23 software package. According to statistical analysis, quantitative data description application (?), using independent sample t test, single factor variance analysis and Pearson correlation analysis, P0.05 difference was statistically significant, with alpha =0.05 as the test level. Results 1 normal endometrium and endometrioid adenocarcinoma tissue m RNA table of SOX17, beta -catenin and Cyclin D1 gene. The results showed that the expression level of SOX17 gene in endometrioid adenocarcinoma was decreased, and the expression of beta -catenin and Cyclin D1 gene in endometrioid adenocarcinoma tissues increased, and the difference was statistically significant compared with the normal endometrium tissue (P0.05). In 30 cases of endometrioid adenocarcinoma tissue markers. In this study, the relative expression of SOX17, beta -catenin and Cyclin D1 gene m RNA expressed in histological grade, lymph node metastasis and FIGO staging were statistically significant (P0.05). The relative expression of beta -catenin and Cyclin D1 in the depth of myometrium infiltration was statistically significant. After the treatment of HEC1A cells 72h with different concentrations of 5-AZA, the difference of cell growth inhibition by MTT was statistically significant (P0.05), and the inhibition of cell growth was most obvious after 72h (IC50=12.033). With the growth of treatment time, the inhibitory effect of 5-AZA on the growth of HEC1A cells was enhanced, and there was a difference. Statistical significance (P0.05); the expression of SOX17 gene in 5-AZA treated endometrioid adenocarcinoma cells was significantly higher than that before treatment (P0.05), while the RNA expression of M RNA in beta -catenin and Cyclin D1 decreased significantly (P0.05) in.3 endometrioid adenocarcinoma tissue The relative expression of M RNA in Clin D1, beta -catenin and SOX17 gene was analyzed by Pearson 22 correlation analysis. It was found that the expression of SOX17 and Cyclin D1 in endometrioid adenocarcinoma tissues was weakly negative correlation (r=-0.353, P0.05). The expression in endometrioid adenocarcinoma is obviously positive correlation (r=0.863, P0.001). Conclusion 1.SOX17 gene may be a potential tumor suppressor, by downregulating the expression of Cyclin D1 and beta -catenin gene to achieve its tumor inhibition, and provide a new way of thinking for the gene therapy of endometrioid adenocarcinoma,.2. demethylation drug 5- AZA can improve the expression of SOX17 gene in endometrioid adenocarcinoma cell lines, and obviously inhibit the proliferation of endometrial carcinoma cells. In the clinical treatment of endometrial cancer, it may become a potential effective therapeutic drug, and it can be used to achieve its anticancer use by acting SOX17 gene.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
本文编号：2134756
[Abstract]:Background and objective endometrial carcinoma is a common malignant tumor in gynecology. In recent years, the incidence of the incidence of endometriosis, especially in young patients, is on the rise. The pathogenesis of endometrial cancer is not clear, and epigenetic changes play an important role in the development of tumor. At present, SOX17 is developing in endometrial cancer. The role of SOX17 is not yet clear. The aim of this study is to explore the relationship between SOX17 and the development of endometrial carcinoma..SOX17 is an important antagonist of the classic Wnt signaling pathway, which regulates the signal transduction of Wnt/ beta -catenin by inhibiting beta -catenin/TCF dependent transcription and cancer cell proliferation and colony formation. Several studies have shown that the high level of SOX17 promoter is high. It is the main reason for the inactivation of SOX17 gene, which shows low rate of expression in the tissue and leads to the proliferation of cancer cells. Other studies have shown that the inhibition of tumor mechanism by SOX17 may be related to the effect of cell stagnation at G1/S stage, inducing apoptosis and regulating the angiogenesis of tumor cells. Thus, SOX17 may be in human tumor suppressor effect. It plays a role, but its potential role in human endometrial cancer has not been elucidated. Human tumor is a cyclical disease, cyclin D1 (Cyclin D1), as a positive regulator of cell cycle, promotes the proliferation of tumor cells. The methyltransferase inhibitor 5- nitrogen heterocytidine (5-AZA) can be induced by demethylation. Our previous study has shown that the expression of SOX17 gene in endometrial carcinoma tissues and cell lines is decreased, and that the promoter is in the state of hypermethylation. This study uses demethylation drug 5-AZA to act on endometrioid adenocarcinoma cells, real-time fluorescence quantitative PCR detection The expression of SOX17 and Cyclin D1 genes before and after drug action, and the expression of SOX17 and Cyclin D1 in endometrioid adenocarcinoma tissue, to explore the role of SOX17 in the development of endometrioid adenocarcinoma and its possible mechanism. Materials and methods 1. experimental material 1.1 cell sources: human endometrioid adenocarcinoma cell line HE C1A differentiation, from the First Affiliated Hospital of the Zhengzhou University, the Key Laboratory of the First Affiliated Hospital of the Zhengzhou University,.1.2 tissue specimen source and case data: collection of endometrium tissue specimens from the Second Affiliated Hospital of Zhengzhou University, July 2014, at the Second Affiliated Hospital of Zhengzhou University. The specimens were harvested immediately after the operation in vitro, and were quickly stored at -80 C. The samples included 30 cases of uterus. Endometrioid adenocarcinoma and 10 cases of normal proliferative endometrium, all specimens were divided into two groups, 30 cases of endometrioid adenocarcinoma, 10 cases of endometrioid endometrium endometrium, and 10 cases of endometrioid endometrium in normal hyperplastic period of endometrium. All specimens were divided into 2.1 groups, which were confirmed by the pathology department of the Second Affiliated Hospital of Zhengzhou University. The specimens were divided into two groups, 30 cases of endometrioid adenocarcinoma, and 10 cases of normal hyperplasia. No radiotherapy, chemotherapy, hormone immunotherapy, and other antitumor treatment were performed before operation, and patients with other tumors were excluded. The tissue specimens were examined by a pathologist to confirm the diagnosis of SOX17, beta -catenin and Cyclin D1 in endometrioid adenocarcinoma tissue of.2.2 and endometrioid adenocarcinoma. The expression of M RNA: the effects of SOX17, beta -catenin and Cyclin D1 on the proliferation of endometrioid adenocarcinoma cells in 30 cases of endometrioid adenocarcinoma and 10 normal endometrium tissues of the above 30 cases of endometrioid adenocarcinoma and 10 cases of normal endometrium were detected by real-time fluorescent quantitative PCR: cell culture and 5-A with different concentrations of 5-A. ZA treatment of endometrioid adenocarcinoma cell line HEC1A was used to detect cell growth inhibition. The effects of 5-AZA on the growth inhibition of HEC1A cells with different concentrations of 24h, 48h, 72h were detected by MTT method. The m RNA table of three genes in HEC1A before and after the real-time fluorescence quantitative PCR detection was used for the logarithmic logarithm of the 23 software package. According to statistical analysis, quantitative data description application (?), using independent sample t test, single factor variance analysis and Pearson correlation analysis, P0.05 difference was statistically significant, with alpha =0.05 as the test level. Results 1 normal endometrium and endometrioid adenocarcinoma tissue m RNA table of SOX17, beta -catenin and Cyclin D1 gene. The results showed that the expression level of SOX17 gene in endometrioid adenocarcinoma was decreased, and the expression of beta -catenin and Cyclin D1 gene in endometrioid adenocarcinoma tissues increased, and the difference was statistically significant compared with the normal endometrium tissue (P0.05). In 30 cases of endometrioid adenocarcinoma tissue markers. In this study, the relative expression of SOX17, beta -catenin and Cyclin D1 gene m RNA expressed in histological grade, lymph node metastasis and FIGO staging were statistically significant (P0.05). The relative expression of beta -catenin and Cyclin D1 in the depth of myometrium infiltration was statistically significant. After the treatment of HEC1A cells 72h with different concentrations of 5-AZA, the difference of cell growth inhibition by MTT was statistically significant (P0.05), and the inhibition of cell growth was most obvious after 72h (IC50=12.033). With the growth of treatment time, the inhibitory effect of 5-AZA on the growth of HEC1A cells was enhanced, and there was a difference. Statistical significance (P0.05); the expression of SOX17 gene in 5-AZA treated endometrioid adenocarcinoma cells was significantly higher than that before treatment (P0.05), while the RNA expression of M RNA in beta -catenin and Cyclin D1 decreased significantly (P0.05) in.3 endometrioid adenocarcinoma tissue The relative expression of M RNA in Clin D1, beta -catenin and SOX17 gene was analyzed by Pearson 22 correlation analysis. It was found that the expression of SOX17 and Cyclin D1 in endometrioid adenocarcinoma tissues was weakly negative correlation (r=-0.353, P0.05). The expression in endometrioid adenocarcinoma is obviously positive correlation (r=0.863, P0.001). Conclusion 1.SOX17 gene may be a potential tumor suppressor, by downregulating the expression of Cyclin D1 and beta -catenin gene to achieve its tumor inhibition, and provide a new way of thinking for the gene therapy of endometrioid adenocarcinoma,.2. demethylation drug 5- AZA can improve the expression of SOX17 gene in endometrioid adenocarcinoma cell lines, and obviously inhibit the proliferation of endometrial carcinoma cells. In the clinical treatment of endometrial cancer, it may become a potential effective therapeutic drug, and it can be used to achieve its anticancer use by acting SOX17 gene.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
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