PPARγ、ERα和ERβ在子宫内膜癌的表达及其调控关系分析
发布时间：2018-09-03 12:35
【摘要】：目的 子宫内膜癌是女性生殖系统最常见的恶性肿瘤之一,其发病率在世界范围内呈上升趋势,甚至在部分发达国家,已成为妇科恶性肿瘤的首位。I型子宫内膜癌被认为是一种雌激素依赖型肿瘤,其发生与无孕激素拮抗的雌激素长期作用相关。目前,肥胖、高血压、糖尿病等因素被视为子宫内膜癌发病的高危因素。然而,以拮抗雌激素受体为基础的激素治疗并非对所有雌激素受体阳性子宫内膜癌患者都有效,推测可能存在雌激素—雌激素受体通路以外的其他信号通路促进或介导子宫内膜癌的发生。近年研究已证实过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ, PPARy)与脂肪细胞分化、肥胖、胰岛素抵抗、糖尿病及肿瘤等多种疾病密切相关,与子宫内膜癌的发生发展也必然有一定关系。鉴于雌激素受体α (estrogen receptor α, ERα)、雌激素受体β(estrogen receptor β,ERβ)和PPARy均属于细胞核受体,核受体之间存在交互作用,在子宫内膜癌领域中关于此三者相互联系的报告较少。因此,本研究通过检测子宫内膜癌组织中PPARγ、ERα和ERβ的表达,分析三者在子宫内膜癌组织中的相互联系,探讨其在子宫内膜癌发生发展中的作用及其相互联系的可能机制。 方法 本研究收集子宫内膜癌标本45例(组织学类型均为腺癌,其中高分化腺癌14例,中分化腺癌17例,低分化腺癌14例),同时选取正常子宫内膜组织标本13例作为对照。采用免疫组化SP法及western blot法检测正常子宫内膜及高中低分化子宫内膜癌组织中PPARγ、ERα和ERβ的表达情况。通过Pearson相关分析PPARγ、 ERα与ERβ表达之间的相关性。同时,我们选取子宫内膜癌细胞系ECC-1及KLE为研究对象,通过分别转染PPARy表达载体及PPARy siRNA调控PPARy的表达后,检测其对ERα、ERβ的表达影响,qRT-PCR及western blot检测转染后mRNA及蛋白水平变化,并通过Transwell体外迁移、侵袭实验检测其细胞迁移、侵袭能力的影响,CCK-8实验检测其对细胞增殖能力的影响,分析PPARy在子宫内膜癌发生发展中的可能作用。继而,我们再次通过转染ERa和ERβ表达载体上调ERa及ERβ的表达,检测ERs对PI3K/AKT/mTOR信号通路的影响,探讨ERs发挥作用的可能机制。实验所得数据应用SPSS17.0统计学软件分析,数据以x±s表示,两组数据比较采用t检验,多组数据比较采用单因素方差分析,小样本数据比较采用非参数秩和检验。Pearson相关分析计算各指标间相关系数。以P0.05表示差异有统计学意义。 结果 1.PPARy在子宫内膜癌中表达量明显降低,且随着病理分级的进展,呈递减趋势(P0.05); ERα在正常子宫内膜及高分化子宫内膜样腺癌中表达量无明显差异(P0.05),而在中分化及、低分化子宫内膜癌中表达量随病理分级的进展逐渐降低(P0.05); ERβ表达量仅在低分化子宫内膜癌组织中明显降低(P0.05),而在正常子宫内膜及高、中分化子宫内膜癌中其表达量无明显差异(P0.05)。 2.通过Pearson相关分析示ERa与PPARy在不同子宫内膜组织中表达量存在明显正相关(P0.05),而ERa与ERβ、ERβ与PPARy间无相关性(P0.05)。 3.上调PPARy的表达后,ERa表达量降低,Transwell体外迁移、侵袭实验示穿过聚碳酸酯膜及Matrigel胶的子宫内膜癌细胞数目减少,CCK-8实验示细胞活力降低,与对照组有明显差异(P0.05),下调PPARy的表达后,ERa表达量增多,Transwell体外迁移、侵袭实验示穿过聚碳酸酯膜及Matrigel胶的细胞数目增多,CCK-8实验示细胞活力增强,与对照组有明显差异(P0.05)。而调控PPARy的表达后对ERβ的表达无明显影响。 4.上调ERa的表达后,western blot结果示PI3K p85α、p-AKT及p-mTOR蛋白表达水平升高,与对照组有明显差异(P0.05)；而ERβ的表达则对此信号通路蛋白表达无明显影响(P0.05)。 结论 LPPARy及ERa的表达水平与子宫内膜癌的分化程度、临床分期相关,PPARy与ERa间存在负性调控关系,PPARy可能通过调控ERa的表达在子宫内膜癌的发生发展中可能起重要作用。 2.上调PPARy的表达后,子宫内膜癌细胞侵袭、增殖能力显著降低；下调PPARy的表达后,子宫内膜癌细胞侵袭、增殖能力显著增强。 3.上调ERa的表达可激活PI3K/AKT/mTOR信号通路,可能为其调控子宫内膜癌细胞侵袭、增殖能力的机制。 4.调控PPARy及ERa的表达,可为子宫内膜癌的治疗提供新的靶点。
[Abstract]:objective
Endometrial carcinoma is one of the most common malignant tumors in the female reproductive system. Its incidence is on the rise in the world. It has become the first gynecological malignant tumor in some developed countries. Currently, obesity, hypertension, diabetes and other factors are considered to be high-risk factors for endometrial cancer. However, estrogen receptor antagonist-based hormone therapy is not effective for all estrogen receptor-positive endometrial cancer patients, suggesting that there may be other signaling pathways other than estrogen-estrogen receptor pathway. Recent studies have confirmed that peroxisome proliferator-activated receptor gamma (PPARy) is closely related to adipocyte differentiation, obesity, insulin resistance, diabetes mellitus and tumors, and is also associated with the development of endometrial cancer. Since estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta) and PARy belong to nuclear receptors, there are few reports about the interaction between them in the field of endometrial carcinoma. The expression of ER-beta and ER-beta in endometrial carcinoma tissues was analyzed to explore their roles in the development of endometrial carcinoma and the possible mechanisms.
Method
In this study, 45 endometrial carcinoma specimens (including 14 well-differentiated adenocarcinoma, 17 moderately differentiated adenocarcinoma and 14 poorly differentiated adenocarcinoma) were collected and 13 normal endometrial tissue specimens were selected as controls. The expression of PPAR-gamma, ER-alpha and ER-beta in endometrial carcinoma cell lines ECC-1 and KLE were detected by Pearson correlation analysis. The expression of PPAR-gamma, ER-alpha and ER-beta was regulated by PPARy expression vector and PPARy siRNA respectively, and the expression of ER-alpha and ER-beta was detected by qRT-PCR. The changes of mRNA and protein levels after transfection were detected by Western blot, and the effects of Transwell on cell migration and invasion were examined by in vitro migration and invasion experiments. CCK-8 assay was used to examine the effects of PPARy on cell proliferation and to analyze the possible role of PPARy in the development of endometrial carcinoma. The expression of ERa and ERbeta was up-regulated by vector, and the effect of ER s on PI3K/AKT/mTOR signaling pathway was detected, and the possible mechanism of ER s was explored. Nonparametric rank sum test. Pearson correlation analysis was used to calculate the correlation coefficients among the indexes. The difference was statistically significant with P 0.05.
Result
1. The expression of PPARy in endometrial carcinoma decreased significantly, and showed a decreasing trend with the progress of pathological grading (P 0.05); the expression of ER alpha in normal endometrium and differentiated endometrioid adenocarcinoma had no significant difference (P 0.05), but in moderately differentiated and poorly differentiated endometrial carcinoma, the expression decreased gradually with the progress of pathological grading (P 0.05). The expression of Rbeta was significantly decreased in poorly differentiated endometrial carcinoma (P 0.05), but not in normal endometrium and moderately differentiated endometrial carcinoma (P 0.05).
2. Pearson correlation analysis showed that there was a significant positive correlation between the expression of ERa and PPARy in different endometrial tissues (P 0.05), but there was no correlation between ERa, ERbeta and PPARy (P 0.05).
3. Up-regulated expression of PPARy decreased the expression of ERa, and Transwell migrated in vitro. Invasion test showed that the number of endometrial cancer cells passing through polycarbonate membrane and Matrigel gum decreased. CCK-8 test showed that cell viability decreased significantly compared with the control group (P 0.05). After down-regulated expression of PPARy, the expression of ERa increased, and Transwell migrated and invaded in vitro. The results showed that the number of cells passing through the polycarbonate membrane and Matrigel gel increased. CCK-8 showed that the cell viability was enhanced, which was significantly different from that of the control group (P 0.05). However, the expression of ER beta was not affected by PPARy regulation.
4. Up-regulated expression of ERa, Western blot showed that PI3K p85a, p-AKT and p-mTOR protein expression levels increased significantly compared with the control group (P 0.05), but the expression of ER beta had no significant effect on the expression of this signaling pathway protein (P 0.05).
conclusion
The expression levels of LPPARy and ERa were correlated with the differentiation degree and clinical stage of endometrial carcinoma. There was a negative relationship between PPARy and ERa. PPARy may play an important role in the occurrence and development of endometrial carcinoma by regulating the expression of ERa.
2. Up-regulation of PPARy expression significantly reduced invasion and proliferation of endometrial cancer cells; down-regulation of PPARy expression significantly increased invasion and proliferation of endometrial cancer cells.
3. Upregulation of ERa expression can activate PI3K/AKT/mTOR signaling pathway, which may be the mechanism of regulating invasion and proliferation of endometrial carcinoma cells.
4. regulation of the expression of PPARy and ERa can provide new targets for the treatment of endometrial cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2219996
[Abstract]:objective
Endometrial carcinoma is one of the most common malignant tumors in the female reproductive system. Its incidence is on the rise in the world. It has become the first gynecological malignant tumor in some developed countries. Currently, obesity, hypertension, diabetes and other factors are considered to be high-risk factors for endometrial cancer. However, estrogen receptor antagonist-based hormone therapy is not effective for all estrogen receptor-positive endometrial cancer patients, suggesting that there may be other signaling pathways other than estrogen-estrogen receptor pathway. Recent studies have confirmed that peroxisome proliferator-activated receptor gamma (PPARy) is closely related to adipocyte differentiation, obesity, insulin resistance, diabetes mellitus and tumors, and is also associated with the development of endometrial cancer. Since estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta) and PARy belong to nuclear receptors, there are few reports about the interaction between them in the field of endometrial carcinoma. The expression of ER-beta and ER-beta in endometrial carcinoma tissues was analyzed to explore their roles in the development of endometrial carcinoma and the possible mechanisms.
Method
In this study, 45 endometrial carcinoma specimens (including 14 well-differentiated adenocarcinoma, 17 moderately differentiated adenocarcinoma and 14 poorly differentiated adenocarcinoma) were collected and 13 normal endometrial tissue specimens were selected as controls. The expression of PPAR-gamma, ER-alpha and ER-beta in endometrial carcinoma cell lines ECC-1 and KLE were detected by Pearson correlation analysis. The expression of PPAR-gamma, ER-alpha and ER-beta was regulated by PPARy expression vector and PPARy siRNA respectively, and the expression of ER-alpha and ER-beta was detected by qRT-PCR. The changes of mRNA and protein levels after transfection were detected by Western blot, and the effects of Transwell on cell migration and invasion were examined by in vitro migration and invasion experiments. CCK-8 assay was used to examine the effects of PPARy on cell proliferation and to analyze the possible role of PPARy in the development of endometrial carcinoma. The expression of ERa and ERbeta was up-regulated by vector, and the effect of ER s on PI3K/AKT/mTOR signaling pathway was detected, and the possible mechanism of ER s was explored. Nonparametric rank sum test. Pearson correlation analysis was used to calculate the correlation coefficients among the indexes. The difference was statistically significant with P 0.05.
Result
1. The expression of PPARy in endometrial carcinoma decreased significantly, and showed a decreasing trend with the progress of pathological grading (P 0.05); the expression of ER alpha in normal endometrium and differentiated endometrioid adenocarcinoma had no significant difference (P 0.05), but in moderately differentiated and poorly differentiated endometrial carcinoma, the expression decreased gradually with the progress of pathological grading (P 0.05). The expression of Rbeta was significantly decreased in poorly differentiated endometrial carcinoma (P 0.05), but not in normal endometrium and moderately differentiated endometrial carcinoma (P 0.05).
2. Pearson correlation analysis showed that there was a significant positive correlation between the expression of ERa and PPARy in different endometrial tissues (P 0.05), but there was no correlation between ERa, ERbeta and PPARy (P 0.05).
3. Up-regulated expression of PPARy decreased the expression of ERa, and Transwell migrated in vitro. Invasion test showed that the number of endometrial cancer cells passing through polycarbonate membrane and Matrigel gum decreased. CCK-8 test showed that cell viability decreased significantly compared with the control group (P 0.05). After down-regulated expression of PPARy, the expression of ERa increased, and Transwell migrated and invaded in vitro. The results showed that the number of cells passing through the polycarbonate membrane and Matrigel gel increased. CCK-8 showed that the cell viability was enhanced, which was significantly different from that of the control group (P 0.05). However, the expression of ER beta was not affected by PPARy regulation.
4. Up-regulated expression of ERa, Western blot showed that PI3K p85a, p-AKT and p-mTOR protein expression levels increased significantly compared with the control group (P 0.05), but the expression of ER beta had no significant effect on the expression of this signaling pathway protein (P 0.05).
conclusion
The expression levels of LPPARy and ERa were correlated with the differentiation degree and clinical stage of endometrial carcinoma. There was a negative relationship between PPARy and ERa. PPARy may play an important role in the occurrence and development of endometrial carcinoma by regulating the expression of ERa.
2. Up-regulation of PPARy expression significantly reduced invasion and proliferation of endometrial cancer cells; down-regulation of PPARy expression significantly increased invasion and proliferation of endometrial cancer cells.
3. Upregulation of ERa expression can activate PI3K/AKT/mTOR signaling pathway, which may be the mechanism of regulating invasion and proliferation of endometrial carcinoma cells.
4. regulation of the expression of PPARy and ERa can provide new targets for the treatment of endometrial cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
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