PPARγ、ERα和ERβ在子宮內(nèi)膜癌的表達及其調(diào)控關(guān)系分析
發(fā)布時間：2018-09-03 12:35
【摘要】：目的 子宮內(nèi)膜癌是女性生殖系統(tǒng)最常見的惡性腫瘤之一,其發(fā)病率在世界范圍內(nèi)呈上升趨勢,甚至在部分發(fā)達國家,已成為婦科惡性腫瘤的首位。I型子宮內(nèi)膜癌被認為是一種雌激素依賴型腫瘤,其發(fā)生與無孕激素拮抗的雌激素長期作用相關(guān)。目前,肥胖、高血壓、糖尿病等因素被視為子宮內(nèi)膜癌發(fā)病的高危因素。然而,以拮抗雌激素受體為基礎(chǔ)的激素治療并非對所有雌激素受體陽性子宮內(nèi)膜癌患者都有效,推測可能存在雌激素—雌激素受體通路以外的其他信號通路促進或介導子宮內(nèi)膜癌的發(fā)生。近年研究已證實過氧化物酶體增殖物激活受體γ(peroxisome proliferator-activated receptor γ, PPARy)與脂肪細胞分化、肥胖、胰島素抵抗、糖尿病及腫瘤等多種疾病密切相關(guān),與子宮內(nèi)膜癌的發(fā)生發(fā)展也必然有一定關(guān)系。鑒于雌激素受體α (estrogen receptor α, ERα)、雌激素受體β(estrogen receptor β,ERβ)和PPARy均屬于細胞核受體,核受體之間存在交互作用,在子宮內(nèi)膜癌領(lǐng)域中關(guān)于此三者相互聯(lián)系的報告較少。因此,本研究通過檢測子宮內(nèi)膜癌組織中PPARγ、ERα和ERβ的表達,分析三者在子宮內(nèi)膜癌組織中的相互聯(lián)系,探討其在子宮內(nèi)膜癌發(fā)生發(fā)展中的作用及其相互聯(lián)系的可能機制。 方法 本研究收集子宮內(nèi)膜癌標本45例(組織學類型均為腺癌,其中高分化腺癌14例,中分化腺癌17例,低分化腺癌14例),同時選取正常子宮內(nèi)膜組織標本13例作為對照。采用免疫組化SP法及western blot法檢測正常子宮內(nèi)膜及高中低分化子宮內(nèi)膜癌組織中PPARγ、ERα和ERβ的表達情況。通過Pearson相關(guān)分析PPARγ、 ERα與ERβ表達之間的相關(guān)性。同時,我們選取子宮內(nèi)膜癌細胞系ECC-1及KLE為研究對象,通過分別轉(zhuǎn)染PPARy表達載體及PPARy siRNA調(diào)控PPARy的表達后,檢測其對ERα、ERβ的表達影響,qRT-PCR及western blot檢測轉(zhuǎn)染后mRNA及蛋白水平變化,并通過Transwell體外遷移、侵襲實驗檢測其細胞遷移、侵襲能力的影響,CCK-8實驗檢測其對細胞增殖能力的影響,分析PPARy在子宮內(nèi)膜癌發(fā)生發(fā)展中的可能作用。繼而,我們再次通過轉(zhuǎn)染ERa和ERβ表達載體上調(diào)ERa及ERβ的表達,檢測ERs對PI3K/AKT/mTOR信號通路的影響,探討ERs發(fā)揮作用的可能機制。實驗所得數(shù)據(jù)應用SPSS17.0統(tǒng)計學軟件分析,數(shù)據(jù)以x±s表示,兩組數(shù)據(jù)比較采用t檢驗,多組數(shù)據(jù)比較采用單因素方差分析,小樣本數(shù)據(jù)比較采用非參數(shù)秩和檢驗。Pearson相關(guān)分析計算各指標間相關(guān)系數(shù)。以P0.05表示差異有統(tǒng)計學意義。 結(jié)果 1.PPARy在子宮內(nèi)膜癌中表達量明顯降低,且隨著病理分級的進展,呈遞減趨勢(P0.05); ERα在正常子宮內(nèi)膜及高分化子宮內(nèi)膜樣腺癌中表達量無明顯差異(P0.05),而在中分化及、低分化子宮內(nèi)膜癌中表達量隨病理分級的進展逐漸降低(P0.05); ERβ表達量僅在低分化子宮內(nèi)膜癌組織中明顯降低(P0.05),而在正常子宮內(nèi)膜及高、中分化子宮內(nèi)膜癌中其表達量無明顯差異(P0.05)。 2.通過Pearson相關(guān)分析示ERa與PPARy在不同子宮內(nèi)膜組織中表達量存在明顯正相關(guān)(P0.05),而ERa與ERβ、ERβ與PPARy間無相關(guān)性(P0.05)。 3.上調(diào)PPARy的表達后,ERa表達量降低,Transwell體外遷移、侵襲實驗示穿過聚碳酸酯膜及Matrigel膠的子宮內(nèi)膜癌細胞數(shù)目減少,CCK-8實驗示細胞活力降低,與對照組有明顯差異(P0.05),下調(diào)PPARy的表達后,ERa表達量增多,Transwell體外遷移、侵襲實驗示穿過聚碳酸酯膜及Matrigel膠的細胞數(shù)目增多,CCK-8實驗示細胞活力增強,與對照組有明顯差異(P0.05)。而調(diào)控PPARy的表達后對ERβ的表達無明顯影響。 4.上調(diào)ERa的表達后,western blot結(jié)果示PI3K p85α、p-AKT及p-mTOR蛋白表達水平升高,與對照組有明顯差異(P0.05)；而ERβ的表達則對此信號通路蛋白表達無明顯影響(P0.05)。 結(jié)論 LPPARy及ERa的表達水平與子宮內(nèi)膜癌的分化程度、臨床分期相關(guān),PPARy與ERa間存在負性調(diào)控關(guān)系,PPARy可能通過調(diào)控ERa的表達在子宮內(nèi)膜癌的發(fā)生發(fā)展中可能起重要作用。 2.上調(diào)PPARy的表達后,子宮內(nèi)膜癌細胞侵襲、增殖能力顯著降低；下調(diào)PPARy的表達后,子宮內(nèi)膜癌細胞侵襲、增殖能力顯著增強。 3.上調(diào)ERa的表達可激活PI3K/AKT/mTOR信號通路,可能為其調(diào)控子宮內(nèi)膜癌細胞侵襲、增殖能力的機制。 4.調(diào)控PPARy及ERa的表達,可為子宮內(nèi)膜癌的治療提供新的靶點。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
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