HER2与FASN交互作用调控卵巢癌恶性表型及其分子机制的研究

发布时间：2018-01-17 15:24

本文关键词：HER2与FASN交互作用调控卵巢癌恶性表型及其分子机制的研究　出处：《东南大学》2015年博士论文　论文类型：学位论文

【摘要】：[目的]卵巢癌是女性生殖系统常见的肿瘤之一,其发病率列居女性生殖系统恶性肿瘤第三位,仅次于子宫颈癌和子宫体癌。但卵巢癌致死率却居妇科恶性肿瘤之首,严重威胁着女性的生命与健康,因此探究卵巢癌的发病分子机制成为妇科肿瘤研究的热点之一。新近研究发现：脂肪酸合酶(Fatty acid synthase, FASN)和人类表皮生长因子受体-2((human epidermal growth factor receptor-2, HER2)在多种肿瘤的发生、发展过程中起重要作用,它们能够单独或联合调控肿瘤的发生、发展,其作用可能通过磷脂酰肌醇3-激酶／蛋白激酶B (Phosphoinositide 3-kinase/Protein kinase B,PI3K/Akt)信号通路介导实现。我们的前期的预实验结果显示：FASN和HER2表达水平与卵巢癌间存在一定的相关性,FASN和HER2交互作用是否通过PI3K/Akt信号通路介导并影响卵巢癌的发生、发展及其机理迄今未见报道。本研究初步探讨FASN和HER2表达与卵巢癌的临床病理及预后相关性,并利用分子生物学相关技术,探究PI3K/Akt信号通路介导FASN和HER2交互作用在卵巢癌的发生、发展过程中的相关性,以期为卵巢癌的诊治寻找新的分子靶点提供实验依据和理论基础。[方法]1.经东南大学附属中大医院伦理学委员会同意,收集东南大学附属中大医院卵巢癌患者的标本进行本实验研究,所有标本均取自术前未经放化疗的患者且经病理学确诊。然后应用组织芯片技术检测FASN和HER2在卵巢癌中的表达,结合患者随访结果分析FASN和HER2的表达与患者生存期、预后的关系。2.应用qPCR和Western Blot技术检测人卵巢癌细胞SKOV3、SKOV3/DDP及A2780中HER2和FASN的表达。3.分别构建HER2和FASN干扰质粒,并对HER2和FASN高表达的A2780卵巢癌细胞株进行转染；qPCR筛选干扰效率高的干扰质粒并建立稳定转染FASN和HER2的细胞株。4. qPCR, Western Blot鉴定稳定转染HER2和FASN干扰质粒的卵巢癌A2780细胞株,并检测其细胞增殖能力、侵袭力以及凋亡变化。同时,进一步检测稳转株中PI3K/Akt信号通路相关蛋白的表达。5.应用PI3K特异性抑制剂(ZSTK474)作用于靶细胞,检测其对PI3K/Akt信号通路活性、HER2和FASN表达的影响,探讨其相关性。6.观察PI3K特异性抑制剂对卵巢癌细胞周期、凋亡、克隆形成能力及侵袭力等生物学行为的影响。[结果]1.人卵巢癌组织中,FASN阳性表达率为70.53%(67/95),FANS阳性表达与肿瘤分级以及FIGO分期间具有明显的相关性(P0.001); HER-2阳性表达率为33.68%(32/95),其阳性表达与患者的年龄、肿瘤组织分型、分级、残余肿瘤及FIGO分级均无显著性差异(P0.05)。两组中,同时高表达FASN和HER-2的卵巢癌患者较高表达FASN和低表达HER-2的卵巢癌患者预后更差。2.人卵巢癌细胞SKOV3、SKOV3/DDP及A2780中HER2和FASN mRNA及蛋白的表达结果显示：A2780中FASN、HER2蛋白的表达均高于SKVO3\DDP和SKVO3,具有统计学差异(P0.05),故选取A2780细胞作为后续研究的靶细胞。3.将干扰效果最佳的HER2和FASN干扰质粒以及对应的空载干扰质粒分别转染入A2780细胞,分别建立稳定转染HER2和FASN干扰质粒的A2780细胞株。与空白组和对照组相比,干扰组的HER2和FASN mRNA表达及蛋白表达明显减低,(P0.05)。干扰A2780细胞HER2和FASN表达可显著抑制肿瘤细胞的克隆形成能力和侵袭能力,同时诱导凋亡蛋白Bax的高表达,与其它对照组相比具有统计学意义(P0.05)。4.A2780细胞中,分别转染HER2和FASN两个干扰质粒后PI3K/Akt信号通路的活性明显受抑制。5. PI3K/Akt信号通路特异性抑制剂(ZSTK474)作用A2780细胞后,明显抑制靶细胞的PI3K/Akt信号通路活性,下调HER2和FASN基因和蛋白表达,与其它对照组相比均具有统计学意义(P0.05)。ZSTK474能够将A2780细胞阻滞在G1期,减少其S期的比例,同时诱导A2780细胞的凋亡,与其它对照组比较均具有统计学意义(P0.05)。A2780细胞的生物学行为同样显示,ZSTK474能够降低A2780细胞的克隆形成率和细胞侵袭能力,与其它对照组比较均具有统计学意义(P0.05)。[结论]1.卵巢癌组织的FASN和HER2同时高表达与卵巢癌患者的生存期、预后具有明显的负相关性。2. RNAi技术成功下调了卵巢癌A2780细胞中HER2和FASN的表达,影响了卵巢癌A2780细胞的恶性生物学行为,而HER2和FASN之间可能存在交互作用,该交互作用可能通过PI3K/Akt信号通路介导。3. PI3K/Akt信号通路特异性抑制剂(ZSTK474)可降低卵巢癌A2780细胞PI3K/Akt信号通路的活性,下调卵巢癌A2780细胞HER2与FASNmRNA和蛋白的表达,抑制卵巢癌A2780细胞的恶性生物学行为。研究结果显示：PI3K/Akt信号通路介导FASN和HER2交互作用可能调控卵巢癌的恶性表型,影响卵巢癌的发生、发展以及患者预后。
[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.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.31

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