SOX17和CyclinD1在子宮內(nèi)膜樣腺癌中的表達(dá)及5-AZA對(duì)其表達(dá)的影響
發(fā)布時(shí)間：2018-07-20 20:58
【摘要】：背景與目的子宮內(nèi)膜癌是婦科常見(jiàn)的惡性腫瘤,近年來(lái)其發(fā)病率特別是年輕患者的發(fā)病率呈上升趨勢(shì)。子宮內(nèi)膜癌的發(fā)病機(jī)制尚不明確,表觀遺傳學(xué)的改變?cè)谀[瘤的發(fā)生發(fā)展過(guò)程中起重要作用。就目前而言,SOX17在子宮內(nèi)膜癌發(fā)生發(fā)展中的作用尚不明確,本研究旨在探討SOX17與子宮內(nèi)膜癌發(fā)生發(fā)展的關(guān)系。SOX17是經(jīng)典Wnt信號(hào)通路的重要拮抗劑,通過(guò)抑制β-catenin/TCF依賴性轉(zhuǎn)錄及癌細(xì)胞增殖和集落的形成,從而調(diào)節(jié)Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)。多項(xiàng)研究表明,SOX17啟動(dòng)子的高甲基化是SOX17基因失活的主要原因,其在組織中呈現(xiàn)低表達(dá)率,進(jìn)而導(dǎo)致癌細(xì)胞的增殖。另有研究表明SOX17的抑制腫瘤機(jī)制可能與其使細(xì)胞停滯在G1/S期,誘導(dǎo)凋亡、調(diào)節(jié)腫瘤細(xì)胞的血管生成的作用有關(guān)。由此得出SOX17可能在人類(lèi)抑癌作用中發(fā)揮作用,但其在人類(lèi)子宮內(nèi)膜癌中的潛在作用尚未闡明。人體腫瘤是一種細(xì)胞周期性疾病,細(xì)胞周期素D1(Cyclin D1)作為細(xì)胞周期的正向調(diào)節(jié)因子,促進(jìn)了腫瘤細(xì)胞的增殖。甲基轉(zhuǎn)移酶抑制劑5-氮雜胞苷(5-AZA)能夠通過(guò)去甲基化作用使過(guò)甲基化的基因重新表達(dá),從而恢復(fù)其抑癌功能。我們的前期研究已表明SOX17基因在子宮內(nèi)膜癌組織及細(xì)胞株中表達(dá)下降,與其啟動(dòng)子處于高甲基化狀態(tài)有關(guān),本研究利用去甲基化藥物5-AZA作用子宮內(nèi)膜樣腺癌細(xì)胞,實(shí)時(shí)熒光定量PCR檢測(cè)藥物作用前后SOX17和Cyclin D1基因的表達(dá),同時(shí)對(duì)子宮內(nèi)膜樣腺癌組織中SOX17和Cyclin D1表達(dá)水平進(jìn)行檢測(cè),旨在探討SOX17在子宮內(nèi)膜樣腺癌的發(fā)生發(fā)展中的作用及其可能機(jī)制。材料與方法1.實(shí)驗(yàn)材料1.1細(xì)胞來(lái)源:人子宮內(nèi)膜樣腺癌細(xì)胞株HEC1A中分化,由鄭州大學(xué)第一附屬醫(yī)院重點(diǎn)實(shí)驗(yàn)室饋贈(zèng)。1.2組織標(biāo)本來(lái)源與病例資料:收集2014年7月~2016年12月于鄭州大學(xué)第二附屬醫(yī)院行子宮切除的子宮內(nèi)膜組織標(biāo)本,術(shù)中標(biāo)本離體后立即取材,迅速置于-80℃保存。所取標(biāo)本包括30例子宮內(nèi)膜樣腺癌組織和10例正常增生期子宮內(nèi)膜組織,所有標(biāo)本均經(jīng)鄭州大學(xué)第二附屬醫(yī)院病理科確診。2.實(shí)驗(yàn)方法2.1實(shí)驗(yàn)分組:所收集標(biāo)本共分兩組,子宮內(nèi)膜樣腺癌組30例,正常增生期子宮內(nèi)膜組10例(因?qū)m頸上皮內(nèi)瘤變手術(shù)切除子宮的子宮內(nèi)膜組織),入組患者術(shù)前均未進(jìn)行放療、化療、激素免疫治療及其他抗腫瘤治療,且排除合并其他部位腫瘤的患者,組織標(biāo)本均由病理科醫(yī)師鏡下觀察確診。2.2正常增生期子宮內(nèi)膜組織和子宮內(nèi)膜樣腺癌組織中SOX17、β-catenin和Cyclin D1基因的m RNA表達(dá)情況:采用實(shí)時(shí)熒光定量PCR技術(shù)對(duì)上述30例子宮內(nèi)膜樣腺癌組織和10例正常增生期子宮內(nèi)膜組織中SOX17、β-catenin和Cyclin D1基因表達(dá)情況進(jìn)行檢測(cè)。2.3 5-AZA對(duì)子宮內(nèi)膜樣腺癌細(xì)胞增殖的影響:采用細(xì)胞培養(yǎng)技術(shù),用不同濃度5-AZA處理子宮內(nèi)膜樣腺癌細(xì)胞株HEC1A,檢測(cè)細(xì)胞生長(zhǎng)抑制情況。使用MTT法檢測(cè)24h、48h、72h不同濃度的5-AZA對(duì)HEC1A細(xì)胞的生長(zhǎng)抑制作用的影響;采用實(shí)時(shí)熒光定量PCR檢測(cè)處理前后HEC1A中三種基因的m RNA表達(dá)情況。3.統(tǒng)計(jì)方法應(yīng)用SPSS 23.0軟件包對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,定量資料描述應(yīng)用(?),組間采用獨(dú)立樣本t檢驗(yàn),單因素方差分析及Pearson相關(guān)分析,以P0.05差異有統(tǒng)計(jì)學(xué)意義,以α=0.05為檢驗(yàn)水準(zhǔn)。結(jié)果1正常子宮內(nèi)膜組織和子宮內(nèi)膜樣腺癌組織中SOX17、β-catenin和Cyclin D1基因的m RNA表達(dá)情況。本實(shí)驗(yàn)檢測(cè)結(jié)果顯示,SOX17基因在子宮內(nèi)膜樣腺癌組織中表達(dá)水平下降,同時(shí)β-catenin和Cyclin D1基因在子宮內(nèi)膜樣腺癌組織中表達(dá)升高,與正常增生期子宮內(nèi)膜組織相比,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。在30例子宮內(nèi)膜樣腺癌組織標(biāo)本中,SOX17、β-catenin和Cyclin D1基因m RNA相對(duì)表達(dá)量在組織學(xué)分級(jí)、淋巴結(jié)轉(zhuǎn)移及FIGO分期中表達(dá)差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。其中β-catenin及Cyclin D1的m RNA相對(duì)表達(dá)量在肌層浸潤(rùn)深度中差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2 5-AZA對(duì)子宮內(nèi)膜樣腺癌細(xì)胞增殖的影響使用不同濃度5-AZA處理HEC1A細(xì)胞72h后,經(jīng)MTT法檢測(cè),細(xì)胞的生長(zhǎng)抑制作用差異具有統(tǒng)計(jì)學(xué)意義(P0.05),且處理72h后對(duì)細(xì)胞生長(zhǎng)作用抑制最明顯(IC50=12.033)。隨著處理時(shí)間的增長(zhǎng),不同濃度5-AZA對(duì)HEC1A細(xì)胞生長(zhǎng)抑制效果增強(qiáng),且差異有統(tǒng)計(jì)學(xué)意義(P0.05);5-AZA處理子宮內(nèi)膜樣腺癌細(xì)胞后SOX17基因表達(dá)較處理前明顯增高(P0.05),同時(shí)β-catenin和Cyclin D1基因的m RNA表達(dá)明顯降低(P0.05)。3子宮內(nèi)膜樣腺癌組織中SOX17和Cyclin D1基因的相關(guān)性分析本文對(duì)30例子宮內(nèi)膜樣腺癌組織中Cyclin D1、β-catenin及SOX17基因的m RNA相對(duì)表達(dá)量進(jìn)行pearson兩兩相關(guān)性分析發(fā)現(xiàn),SOX17及Cyclin D1在子宮內(nèi)膜樣腺癌組織中的表達(dá)呈弱負(fù)相關(guān)(r=-0.353,P0.05),SOX17與β-catenin在子宮內(nèi)膜樣腺癌組織中表達(dá)呈負(fù)相關(guān)(r=-0.463,P0.05),β-catenin和Cyclin D1的表達(dá)在子宮內(nèi)膜樣腺癌組織中呈明顯正相關(guān)(r=0.863,P0.001)。結(jié)論1.SOX17基因可能作為一種潛在的腫瘤抑制因子,通過(guò)下調(diào)Cyclin D1及β-catenin基因的表達(dá)來(lái)實(shí)現(xiàn)其腫瘤抑制作用,為子宮內(nèi)膜樣腺癌的基因治療提供新的思路。2.去甲基化藥物5-AZA可提高SOX17基因在子宮內(nèi)膜樣腺癌細(xì)胞株中的表達(dá),并明顯抑制子宮內(nèi)膜癌細(xì)胞的增殖,在未來(lái)子宮內(nèi)膜癌的臨床治療中,其可能成為潛在有效的治療藥物,通過(guò)作用SOX17基因來(lái)實(shí)現(xiàn)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.33
本文編號(hào)：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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.33
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