本文關(guān)鍵詞：HER2與FASN交互作用調(diào)控卵巢癌惡性表型及其分子機制的研究　出處：《東南大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HER2 FASN PI3K/Akt信號通路 卵巢癌 凋亡 增殖 遷移

【摘要】：[目的]卵巢癌是女性生殖系統(tǒng)常見的腫瘤之一,其發(fā)病率列居女性生殖系統(tǒng)惡性腫瘤第三位,僅次于子宮頸癌和子宮體癌。但卵巢癌致死率卻居婦科惡性腫瘤之首,嚴重威脅著女性的生命與健康,因此探究卵巢癌的發(fā)病分子機制成為婦科腫瘤研究的熱點之一。新近研究發(fā)現(xiàn)：脂肪酸合酶(Fatty acid synthase, FASN)和人類表皮生長因子受體-2((human epidermal growth factor receptor-2, HER2)在多種腫瘤的發(fā)生、發(fā)展過程中起重要作用,它們能夠單獨或聯(lián)合調(diào)控腫瘤的發(fā)生、發(fā)展,其作用可能通過磷脂酰肌醇3-激酶／蛋白激酶B (Phosphoinositide 3-kinase/Protein kinase B,PI3K/Akt)信號通路介導(dǎo)實現(xiàn)。我們的前期的預(yù)實驗結(jié)果顯示：FASN和HER2表達水平與卵巢癌間存在一定的相關(guān)性,FASN和HER2交互作用是否通過PI3K/Akt信號通路介導(dǎo)并影響卵巢癌的發(fā)生、發(fā)展及其機理迄今未見報道。本研究初步探討FASN和HER2表達與卵巢癌的臨床病理及預(yù)后相關(guān)性,并利用分子生物學(xué)相關(guān)技術(shù),探究PI3K/Akt信號通路介導(dǎo)FASN和HER2交互作用在卵巢癌的發(fā)生、發(fā)展過程中的相關(guān)性,以期為卵巢癌的診治尋找新的分子靶點提供實驗依據(jù)和理論基礎(chǔ)。[方法]1.經(jīng)東南大學(xué)附屬中大醫(yī)院倫理學(xué)委員會同意,收集東南大學(xué)附屬中大醫(yī)院卵巢癌患者的標本進行本實驗研究,所有標本均取自術(shù)前未經(jīng)放化療的患者且經(jīng)病理學(xué)確診。然后應(yīng)用組織芯片技術(shù)檢測FASN和HER2在卵巢癌中的表達,結(jié)合患者隨訪結(jié)果分析FASN和HER2的表達與患者生存期、預(yù)后的關(guān)系。2.應(yīng)用qPCR和Western Blot技術(shù)檢測人卵巢癌細胞SKOV3、SKOV3/DDP及A2780中HER2和FASN的表達。3.分別構(gòu)建HER2和FASN干擾質(zhì)粒,并對HER2和FASN高表達的A2780卵巢癌細胞株進行轉(zhuǎn)染；qPCR篩選干擾效率高的干擾質(zhì)粒并建立穩(wěn)定轉(zhuǎn)染FASN和HER2的細胞株。4. qPCR, Western Blot鑒定穩(wěn)定轉(zhuǎn)染HER2和FASN干擾質(zhì)粒的卵巢癌A2780細胞株,并檢測其細胞增殖能力、侵襲力以及凋亡變化。同時,進一步檢測穩(wěn)轉(zhuǎn)株中PI3K/Akt信號通路相關(guān)蛋白的表達。5.應(yīng)用PI3K特異性抑制劑(ZSTK474)作用于靶細胞,檢測其對PI3K/Akt信號通路活性、HER2和FASN表達的影響,探討其相關(guān)性。6.觀察PI3K特異性抑制劑對卵巢癌細胞周期、凋亡、克隆形成能力及侵襲力等生物學(xué)行為的影響。[結(jié)果]1.人卵巢癌組織中,FASN陽性表達率為70.53%(67/95),FANS陽性表達與腫瘤分級以及FIGO分期間具有明顯的相關(guān)性(P0.001); HER-2陽性表達率為33.68%(32/95),其陽性表達與患者的年齡、腫瘤組織分型、分級、殘余腫瘤及FIGO分級均無顯著性差異(P0.05)。兩組中,同時高表達FASN和HER-2的卵巢癌患者較高表達FASN和低表達HER-2的卵巢癌患者預(yù)后更差。2.人卵巢癌細胞SKOV3、SKOV3/DDP及A2780中HER2和FASN mRNA及蛋白的表達結(jié)果顯示：A2780中FASN、HER2蛋白的表達均高于SKVO3\DDP和SKVO3,具有統(tǒng)計學(xué)差異(P0.05),故選取A2780細胞作為后續(xù)研究的靶細胞。3.將干擾效果最佳的HER2和FASN干擾質(zhì)粒以及對應(yīng)的空載干擾質(zhì)粒分別轉(zhuǎn)染入A2780細胞,分別建立穩(wěn)定轉(zhuǎn)染HER2和FASN干擾質(zhì)粒的A2780細胞株。與空白組和對照組相比,干擾組的HER2和FASN mRNA表達及蛋白表達明顯減低,(P0.05)。干擾A2780細胞HER2和FASN表達可顯著抑制腫瘤細胞的克隆形成能力和侵襲能力,同時誘導(dǎo)凋亡蛋白Bax的高表達,與其它對照組相比具有統(tǒng)計學(xué)意義(P0.05)。4.A2780細胞中,分別轉(zhuǎn)染HER2和FASN兩個干擾質(zhì)粒后PI3K/Akt信號通路的活性明顯受抑制。5. PI3K/Akt信號通路特異性抑制劑(ZSTK474)作用A2780細胞后,明顯抑制靶細胞的PI3K/Akt信號通路活性,下調(diào)HER2和FASN基因和蛋白表達,與其它對照組相比均具有統(tǒng)計學(xué)意義(P0.05)。ZSTK474能夠?qū)2780細胞阻滯在G1期,減少其S期的比例,同時誘導(dǎo)A2780細胞的凋亡,與其它對照組比較均具有統(tǒng)計學(xué)意義(P0.05)。A2780細胞的生物學(xué)行為同樣顯示,ZSTK474能夠降低A2780細胞的克隆形成率和細胞侵襲能力,與其它對照組比較均具有統(tǒng)計學(xué)意義(P0.05)。[結(jié)論]1.卵巢癌組織的FASN和HER2同時高表達與卵巢癌患者的生存期、預(yù)后具有明顯的負相關(guān)性。2. RNAi技術(shù)成功下調(diào)了卵巢癌A2780細胞中HER2和FASN的表達,影響了卵巢癌A2780細胞的惡性生物學(xué)行為,而HER2和FASN之間可能存在交互作用,該交互作用可能通過PI3K/Akt信號通路介導(dǎo)。3. PI3K/Akt信號通路特異性抑制劑(ZSTK474)可降低卵巢癌A2780細胞PI3K/Akt信號通路的活性,下調(diào)卵巢癌A2780細胞HER2與FASNmRNA和蛋白的表達,抑制卵巢癌A2780細胞的惡性生物學(xué)行為。研究結(jié)果顯示：PI3K/Akt信號通路介導(dǎo)FASN和HER2交互作用可能調(diào)控卵巢癌的惡性表型,影響卵巢癌的發(fā)生、發(fā)展以及患者預(yù)后。
[Abstract]:[Objective] ovarian cancer is one of the most common tumor of the female reproductive system, the incidence of malignant tumors of female genital system ranks the third, second only to cervical cancer and uterine cancer. But the mortality rate of ovarian cancer is the first in gynecological malignant tumor, a serious threat to women's lives and health, therefore to explore the molecular pathogenesis of ovarian cancer has become one of the hot research of gynecological tumor. A recent study found that fatty acid synthase (Fatty acid, synthase, FASN) and human epidermal growth factor receptor -2 (human epidermal growth factor (receptor-2, HER2) in a variety of tumor, plays an important role in the development process, they can be alone or in combination with the regulation of tumor occurrence, development, its role may be through the 3- kinase / protein kinase, phosphatidylinositol B (Phosphoinositide 3-kinase/Protein kinase B, PI3K/Akt) signal pathway mediated by our pre implementation. The experimental results show that: there is a correlation between FASN and HER2 expression in ovarian cancer and between FASN and HER2 interaction is mediated through PI3K/Akt signaling pathway and affect the development of ovarian cancer, and its mechanism has not been reported. This study was to explore the clinical pathological features and prognosis between FASN and HER2 expression in ovarian cancer, and the use of the related techniques of molecular biology, to explore PI3K/Akt signaling pathway mediated FASN and HER2 interaction in the development of ovarian cancer, the correlation of the development process, in order to provide experimental basis and theoretical basis. Methods]1. by Zhongda Hospital Affiliated to Southeast University ethics committee approval for the diagnosis and treatment of ovarian cancer to find new molecular targets, ovarian cancer specimens were collected in Zhongda Hospital Affiliated to Southeast University this experiment, all specimens were taken from without preoperative chemotherapy patients and confirmed by pathology. The expression of the application of tissue microarray technology for detection of FASN and HER2 in ovarian cancer, the combination of FASN and HER2 expression and survival analysis of patients with follow-up results, the relationship between the prognosis of.2. and Western application of qPCR Blot detection of human ovarian cancer cell line SKOV3, the expression of.3. HER2 and FASN SKOV3/DDP and A2780 respectively in the construction of HER2 and FASN interference plasmid A2780, ovarian cancer cell lines HER2 and FASN and the high expression of the transfected cell line.4.; qPCR interference plasmid qPCR interference screening high efficiency and establish a stable transfection of FASN and HER2, Western Blot HER2 and identification of stable transfection of FASN plasmid in ovarian cancer cell line A2780, and detect the ability of cell proliferation, invasion stress and apoptosis. At the same time, further detection of stable PI3K/Akt signaling pathway related protein expression of.5. strains using PI3K specific inhibitor (ZSTK474) into target cells, the detection of PI3K/Ak The activity of T signaling pathway, the expression of HER2 and FASN, and to explore the relationship between.6. observation of PI3K specific inhibitors on cell cycle and apoptosis of ovarian cancer. The results, influence the colony forming ability and invasiveness of the biological behavior of]1. in human ovarian carcinoma, the positive expression rate of FASN was 70.53% (67/95), the positive expression of FANS and tumor grade and FIGO stage were significantly correlated (P0.001); the positive expression rate of HER-2 was 33.68% (32/95), the positive expression and patient age, tumor type, grade, there were no significant differences between the residual tumor and FIGO classification (P0.05). The two group, and high expression in patients with ovarian cancer prognosis of ovarian cancer patients FASN and HER-2 high expression of FASN and low expression of HER-2 had worse.2. human ovarian cancer cell line SKOV3, the expression of SKOV3/DDP and A2780 in HER2 and FASN mRNA and protein showed that A2780 in FASN, the expression of HER2 protein was higher than that of SKVO3 DDP and SKVO3, with statistical difference (P0.05), the A2780 cells were used as target cells for further research will.3. the best effect of interference HER2 and FASN interference plasmid and the corresponding empty plasmid were transfected into A2780 cells, respectively, to establish a stable transfection of HER2 and FASN interference plasmid in A2780 cell line. Compared with the blank group and the the control group, the expression of HER2 and FASN interference and the expression of mRNA protein was significantly decreased (P0.05). The expression of HER2 and FASN interference A2780 cell clones could significantly inhibit the formation of tumor cells and the invasion ability, high expression of apoptosis protein Bax, compared with the other groups with statistical significance (P0.05) in.4.A2780 cells HER2 and FASN, were transfected into two plasmid after PI3K/Akt pathway was significantly affected by the.5. PI3K/Akt signaling pathway specific inhibitor (ZSTK474) in A2780 cells, inhibit PI3K/Akt signaling pathway activity of target cells, down regulate the expression of HER2 and FASN gene and protein, compared with the other groups were statistically significant (P0.05).ZSTK474 to A2780 cell arrest in G1 phase, decrease the ratio of S phase, and apoptosis induced by A2780 cells, and other groups were statistically significant (P0.05 the biological behavior of.A2780 cells) also shows that ZSTK474 can reduce the formation rate of A2780 cells and cell invasion, and other groups were statistically significant (P0.05). Conclusion]1. ovarian cancer tissue FASN and HER2 simultaneously with high expression in patients with ovarian cancer survival, the prognosis is a negative correlation between.2. RNAi significantly the success of down regulated the expression of HER2 and FASN in ovarian cancer cell line A2780, affecting the malignant biological behavior of ovarian cancer A2780 cells, which may exist between HER2 and FASN interaction, the The interaction may be mediated through PI3K/Akt signaling pathway.3. PI3K/Akt signaling pathway inhibitor (ZSTK474) can reduce the ovarian cancer cell line A2780 PI3K/Akt pathway, expression of ovarian cancer cell line A2780 and HER2 FASNmRNA and protein, inhibit the malignant biological behavior of ovarian cancer A2780 cells. The results showed that PI3K/Akt signaling pathway mediated by FASN and the HER2 interaction may control malignant phenotype of ovarian cancer, affect ovarian cancer, development and prognosis of the patients.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.31

【相似文獻】

相關(guān)期刊論文 前1條

1 張松峰;田華;潘立新;;FASN在直腸癌患者外周血中的表達變化及意義[J];中國普通外科雜志;2013年10期

相關(guān)博士學(xué)位論文 前1條

1 蔡云朗;HER2與FASN交互作用調(diào)控卵巢癌惡性表型及其分子機制的研究[D];東南大學(xué);2015年

相關(guān)碩士學(xué)位論文 前1條

1 馮奇;日糧NDF水平對斷奶獺兔腸MUC1、MUC2與FASN表達的影響[D];西北農(nóng)林科技大學(xué);2013年

，

本文編號：1436832