PPARγ、ERα和ERβ在子宮內(nèi)膜癌的表達(dá)及其調(diào)控關(guān)系分析
發(fā)布時(shí)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(jiàn)的惡性腫瘤之一,其發(fā)病率在世界范圍內(nèi)呈上升趨勢(shì),甚至在部分發(fā)達(dá)國(guó)家,已成為婦科惡性腫瘤的首位。I型子宮內(nèi)膜癌被認(rèn)為是一種雌激素依賴型腫瘤,其發(fā)生與無(wú)孕激素拮抗的雌激素長(zhǎng)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(duì)所有雌激素受體陽(yáng)性子宮內(nèi)膜癌患者都有效,推測(cè)可能存在雌激素—雌激素受體通路以外的其他信號(hào)通路促進(jìn)或介導(dǎo)子宮內(nèi)膜癌的發(fā)生。近年研究已證實(shí)過(guò)氧化物酶體增殖物激活受體γ(peroxisome proliferator-activated receptor γ, PPARy)與脂肪細(xì)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(xì)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(bào)告較少。因此,本研究通過(guò)檢測(cè)子宮內(nèi)膜癌組織中PPARγ、ERα和ERβ的表達(dá),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(jī)制。 方法 本研究收集子宮內(nèi)膜癌標(biāo)本45例(組織學(xué)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(shí)選取正常子宮內(nèi)膜組織標(biāo)本13例作為對(duì)照。采用免疫組化SP法及western blot法檢測(cè)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(dá)情況。通過(guò)Pearson相關(guān)分析PPARγ、 ERα與ERβ表達(dá)之間的相關(guān)性。同時(shí),我們選取子宮內(nèi)膜癌細(xì)胞系ECC-1及KLE為研究對(duì)象,通過(guò)分別轉(zhuǎn)染PPARy表達(dá)載體及PPARy siRNA調(diào)控PPARy的表達(dá)后,檢測(cè)其對(duì)ERα、ERβ的表達(dá)影響,qRT-PCR及western blot檢測(cè)轉(zhuǎn)染后mRNA及蛋白水平變化,并通過(guò)Transwell體外遷移、侵襲實(shí)驗(yàn)檢測(cè)其細(xì)胞遷移、侵襲能力的影響,CCK-8實(shí)驗(yàn)檢測(cè)其對(duì)細(xì)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?cè)俅瓮ㄟ^(guò)轉(zhuǎn)染ERa和ERβ表達(dá)載體上調(diào)ERa及ERβ的表達(dá),檢測(cè)ERs對(duì)PI3K/AKT/mTOR信號(hào)通路的影響,探討ERs發(fā)揮作用的可能機(jī)制。實(shí)驗(yàn)所得數(shù)據(jù)應(yīng)用SPSS17.0統(tǒng)計(jì)學(xué)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(yàn),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(yàn)。Pearson相關(guān)分析計(jì)算各指標(biāo)間相關(guān)系數(shù)。以P0.05表示差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.PPARy在子宮內(nèi)膜癌中表達(dá)量明顯降低,且隨著病理分級(jí)的進(jìn)展,呈遞減趨勢(shì)(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(dá)量無(wú)明顯差異(P0.05),而在中分化及、低分化子宮內(nèi)膜癌中表達(dá)量隨病理分級(jí)的進(jìn)展逐漸降低(P0.05); ERβ表達(dá)量?jī)H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(dá)量無(wú)明顯差異(P0.05)。 2.通過(guò)Pearson相關(guān)分析示ERa與PPARy在不同子宮內(nèi)膜組織中表達(dá)量存在明顯正相關(guān)(P0.05),而ERa與ERβ、ERβ與PPARy間無(wú)相關(guān)性(P0.05)。 3.上調(diào)PPARy的表達(dá)后,ERa表達(dá)量降低,Transwell體外遷移、侵襲實(shí)驗(yàn)示穿過(guò)聚碳酸酯膜及Matrigel膠的子宮內(nèi)膜癌細(xì)胞數(shù)目減少,CCK-8實(shí)驗(yàn)示細(xì)胞活力降低,與對(duì)照組有明顯差異(P0.05),下調(diào)PPARy的表達(dá)后,ERa表達(dá)量增多,Transwell體外遷移、侵襲實(shí)驗(yàn)示穿過(guò)聚碳酸酯膜及Matrigel膠的細(xì)胞數(shù)目增多,CCK-8實(shí)驗(yàn)示細(xì)胞活力增強(qiáng),與對(duì)照組有明顯差異(P0.05)。而調(diào)控PPARy的表達(dá)后對(duì)ERβ的表達(dá)無(wú)明顯影響。 4.上調(diào)ERa的表達(dá)后,western blot結(jié)果示PI3K p85α、p-AKT及p-mTOR蛋白表達(dá)水平升高,與對(duì)照組有明顯差異(P0.05)；而ERβ的表達(dá)則對(duì)此信號(hào)通路蛋白表達(dá)無(wú)明顯影響(P0.05)。 結(jié)論 LPPARy及ERa的表達(dá)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(fù)性調(diào)控關(guān)系,PPARy可能通過(guò)調(diào)控ERa的表達(dá)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(dá)后,子宮內(nèi)膜癌細(xì)胞侵襲、增殖能力顯著降低；下調(diào)PPARy的表達(dá)后,子宮內(nèi)膜癌細(xì)胞侵襲、增殖能力顯著增強(qiáng)。 3.上調(diào)ERa的表達(dá)可激活PI3K/AKT/mTOR信號(hào)通路,可能為其調(diào)控子宮內(nèi)膜癌細(xì)胞侵襲、增殖能力的機(jī)制。 4.調(diào)控PPARy及ERa的表達(dá),可為子宮內(nèi)膜癌的治療提供新的靶點(diǎn)。
[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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33
本文編號(hào)：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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33
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