FKBP14对上皮性卵巢癌细胞增殖和迁移能力的影响

发布时间：2018-01-16 09:45

本文关键词：FKBP14对上皮性卵巢癌细胞增殖和迁移能力的影响　出处：《南京医科大学》2016年博士论文　论文类型：学位论文

【摘要】：卵巢癌是最常见的妇科恶性肿瘤,具有极高的病死率。绝大多数的卵巢癌是上皮性来源(Epithelial Ovarian Cancer,EOC),常因其高侵袭性和化疗耐药性导致患者预后不良。近年来,虽然手术技术不断改进,化疗药物不断更新,但总体上上皮性卵巢癌的5年生存率并未得到显著提升,因此研究卵巢癌特别是上皮性卵巢癌的发生、发展及浸润转移的相关分子机制,有助于寻找合理有效的治疗靶点,是提高卵巢癌生存率、延长生存期的关键。FKBP14属于FK506结合蛋白家族(FK506-binding proteins,FKBPs),其是免疫抑制剂FK506和雷帕霉素的细胞内受体,具有PPIase(peptidyl-prolyl cis-trans isomerase肽脯氨酰顺反异构酶)活性。近年来的研究发现分布广泛,含量丰富的FKBPs家族成员具有多样的生物学作用,包括：抑制T细胞的活化、信号调节、细胞内钙离子的释放、甾体激素受体复合物的形成以及细胞周期抑制等。例如FKBP12是调控细胞周期和维持细胞内钙稳态的生理因子,而FKBP38是细胞凋亡的负向调控因子。一项近期研究发现,FKBP14在果蝇发育中调节Presenilin蛋白水平和Notch信号通路。FKBP14基因突变在人类会导致一种特殊类型结缔组织病Ehlers-Danlos syndrome(EDS),而受到广泛关注。FKBP14基因突变患者皮肤成纤维细胞内质网池肿大,而在体外实验中,来自FKBP14缺失患者的成纤维细胞,体外培养发现多种细胞外基质(ECM)成分的改变：胶原Ⅰ,Ⅲ,Ⅵ,细胞粘合素(tenascin, TN),纤连蛋白(fibronectin FN),β1整合素(integrins)以及血小板反应蛋白(thrombospondin)明显减少甚至缺失。在肿瘤研究中,FKBPs家族成员多样化的生理作用为探讨肿瘤细胞异常增殖、侵袭转移途径、以及血管形成机制等提供了新思路。目前国内外已有较多FKBPs在肿瘤组织中表达的相关研究报道：FKBP11和FKBP52在肝细胞性肝癌中过表达,FKBP5在前列腺癌、黑色素瘤、神经胶质细胞瘤中过表达。FKBP65在高级别卵巢浆液性癌中表达下降。Vougioukas VI等的研究发现,FKBP14作为EGFR信号通路蛋白之一,参与调控恶性神经胶质细胞瘤(GBM)对抗肿瘤药物Erlotinib的敏感性。虽然目前尚缺乏FKBP14在人类肿瘤细胞中表达的直接研究,但细胞外基质(ECM)的合成、分布及降解与恶性肿瘤的增殖、侵袭密切相关已经得到广泛共识及验证,Notch信号通路的异常激活或结构性活化也与多种组织恶性肿瘤的发病相关,而先前研究发现的FKBP14对细胞外基质(ECM)合成的影响、以及对Notch信号通路的调控作用,让我们有理由推测FKBP14可能参与了肿瘤的发生发展过程。在本研究中,我们检测了FKBP14在上皮性卵巢癌组织中的表达。同时观察靶向沉默FKBP14表达后对卵巢癌细胞增殖、细胞周期和凋亡、迁移侵袭等生物学特征的影响,并探讨其可能的作用机制。本研究提示在上皮性卵巢癌中FKBP14可能是一种癌基因,并可能成为潜在的治疗靶点。第一部分FKBP14在上皮性卵巢癌组织中的表达及其意义研究目的：检测卵巢癌组织及非癌组织中FKBP14的表达,探讨FKBP14与卵巢癌的相关性。研究方法：1.采用RT-PCR方法检测40例卵巢癌组织和40例非癌组织中FKBP14 mRNA的表达,分析其表达是否存在差异。2.采用免疫组化SP方法检测60例不同病理类型上皮性卵巢癌组织中FKBP14蛋白的表达情况,以20例卵巢上皮性良性囊肿组织为对照,分析FKBP14与上皮性卵巢癌之间的相关性。结果：上皮性卵巢癌组织中FKBP14 mRNA及蛋白表达水平均比对照组显著升高,这种表达差异提示FKBP14可能是上皮性卵巢癌的一个癌基因。结论：FKBP14可能参与了上皮性卵巢癌的发生发展过程。第二部分干扰抑制FKBP14的表达对卵巢癌细胞系增殖和迁移能力的影响研究目的：利用RNA干扰技术靶向抑制卵巢癌细胞系SKOV3和H08910中FKBP14的表达,观察基因沉默后对卵巢癌细胞系生物学行为：生长增殖、凋亡和侵袭转移等能力的影响,探讨FKBP14在卵巢癌发病机制中的作用,为卵巢癌的分子靶向治疗提供理论依据。研究方法：1.利用Real-time PCR和Western blot方法检测五个卵巢癌细胞系：A2780,OVCAR3, SKOV3,3A0及H08910中FKBP14蛋白的表达情况,筛选出2个高表达FKBP14基因的上皮性卵巢癌细胞系(SKOV3和HO-8910)。2.构建FKBP14基因重组慢病毒干扰载体：采用全基因化学法合成小干扰siRNA FKBP14靶向序列(GACCACTTTCACTGATTAT)及非沉默序列(CCTAAGGTTAAGTCGCCCTCG),折叠成shRNA后,克隆至PLKO.1逆转录病毒载体上,并通过脂质体Lipofectamine 2000转染HEK293细胞。培养48小时后,收集逆转病毒稳转靶细胞,以亲本细胞和空载体转染细胞作为对照。3.应用CCK8实验检测干扰组(RNAi)、空白对照组(WT)、非干扰序列组(NC)卵巢癌细胞系的增殖能力变化。4. Annexin V-FITC/PI双染法流式细胞仪检测干扰组(RNAi)、空白对照组(WT)、非干扰序列组(NC)卵巢癌细胞系的细胞周期及凋亡变化。5.应用Transwell小室技术,检测干扰组(RNAi)、空白对照组(WT)、非干扰序列组(NC)卵巢癌细胞系迁移和侵袭能力的变化。6.通过Real-time PCR和Western blot技术检测干扰组(RNAi)、空白对照组(WT)、非干扰序列组(NC)卵巢癌细胞系相关通路蛋白的表达：E-cadherin, Twistl,MMP2, BCL-2, BAX, capspase3, PCNA等。7. SPSS 13.0软件进行数据分析,计量资料用平均值±标准差表示,组间差异用独立样本t检验分析。结果：1.干扰组(RNAi)卵巢癌细胞中FKBP14蛋白表达与空白对照组(WT)和非干扰序列组(NC)相比下降了90%,而WT组和NC组间FKBP14表达水平没有差异。2.NC组和WT组间细胞增殖差异没有统计意义,提示干扰shRNA'慢病毒转染系统对卵巢癌细胞没有细胞毒作用。FKBP14干扰组细胞SKOV3和H08910在转染后48小时,72小时都表现出明显增殖抑制。3.FKBP14 shRNA转染后,对比WT组和NC组,RNAi组表现出G0/G1期比例增加,S期比例减少。RNAi组细胞凋亡率比NC组升高9倍。而细胞周期和凋亡情况在NC组和WT组间无差异。4.对比WT组和NC组,RNAi组中细胞增殖相关蛋白PCNA、抗凋亡蛋白Bcl-2表达显著下降,凋亡标志蛋白Caspase3和促凋亡蛋白Bax表达上升。在FKBP14表达抑制后,Bax/Bcl-2比率显著上升。5.细胞侵袭迁移实验中,RNAi组细胞侵袭、迁移率均低于WT组和NC组细胞。6.RNAi组基质金属蛋白酶MMP2和EMT诱导因子Twist表达均低于WT组和NC组,而上皮性标志物E-cadherin表达高于WT组和NC组。结论：1.FKBP14参与了卵巢癌细胞的增殖、凋亡、迁移和侵袭的过程,沉默其表达能显著降低细胞增殖、促进细胞凋亡以及减弱其侵袭迁移能力。提示FKBP14可能是卵巢癌发生发展中的一个癌基因,暗示我们FKBP14有可能成为卵巢癌基因治疗的潜在靶点。2. FKBP14 shRNA通过GO/G1细胞周期捕获抑制细胞增殖,FKBP14通过调控Bax/Bcl-2比率抑制细胞凋亡。3. FKBP14促进卵巢癌细胞侵袭和转移的机制可能与调控上皮细胞间质化(EMT)过程以及影响细胞外基质(ECM)成分有关。
[Abstract]:Ovarian cancer is the most common gynecologic malignant tumor, the mortality rate is high. The majority of ovarian cancer is epithelial origin (Epithelial Ovarian Cancer, EOC), often because of its high invasiveness and resistance to chemotherapy leads to poor prognosis. In recent years, although the surgical technique of continuous improvement, chemotherapy drugs constantly updated, but overall on the epithelial ovarian cancer 5 years survival rate has not improved significantly, so the study of ovarian cancer is epithelial ovarian cancer occurrence, development and metastasis related molecular mechanism, can help to find reasonable and effective therapeutic targets, is to improve the survival rate of ovarian cancer, the key to prolong the survival of.FKBP14 belongs to the FK506 binding protein family (FK506-binding proteins, FKBPs), which is immune inhibitor FK506 and rapamycin intracellular receptors, with PPIase (peptidyl-prolyl cis-trans isomerase peptide prolyl isomerase activity). The study found that in recent years are widely distributed, rich members of the FKBPs family with a variety of biological functions, including: activation, inhibition of T cell signaling, intracellular calcium release, the formation of steroid hormone receptor complexes and cell cycle inhibition. For example, FKBP12 is the control of cell cycle and maintain intracellular physiological factor calcium homeostasis, cell apoptosis and FKBP38 is a negative regulatory factor. A recent study found that FKBP14 in Drosophila development in regulating the protein level of Presenilin and Notch signaling pathway in human.FKBP14 gene mutation leads to a special type of connective tissue disease Ehlers-Danlos syndrome (EDS), has received widespread attention in patients with.FKBP14 mutation into the skin fiber cell endoplasmic reticulum enlargement, in vitro, fibroblasts from FKBP14 deficient patients, found a variety of extracellular matrix in vitro (ECM) The changes of the composition of collagen I, III, VI, cell adhesion factor (tenascin, TN), fibronectin (fibronectin FN), integrin beta 1 (integrins) and thrombospondin (thrombospondin) significantly reduced or absent. In cancer research, the physiological role of FKBPs family members in order to explore the variety of abnormal proliferation tumor cell invasion and metastasis, pathway, provides a new idea and mechanism of angiogenesis. Relevant research reports at home and abroad are more FKBPs in the tumor tissues: FKBP11 and FKBP52 expression in hepatocellular carcinoma, FKBP5 in prostate cancer, melanoma, glioma in overexpression decreased the expression of.Vougioukas VI.FKBP65 in high grade ovarian serous carcinoma, FKBP14 as one of the EGFR signaling protein involved in the regulation of malignant glioma (GBM) antitumor drug sensitive Erlotinib . although there is a lack of direct research on the expression of FKBP14 in human tumor cells, but the extracellular matrix (ECM) synthesis, distribution and degradation and proliferation of malignant tumor, is closely related to the invasion has been a broad consensus and verify the pathogenesis of abnormal activation of Notch pathway activation and structure or tumors the previous studies found that FKBP14 on extracellular matrix (ECM) synthesis, and the Notch signaling pathway regulation, so we have reason to speculate that FKBP14 may be involved in the process of tumor development. In this study, we examined the expression of FKBP14 in epithelial ovarian cancer tissues also observed at the same time. Targeted expression of FKBP14 silencing on ovarian cancer cell proliferation, cell cycle and apoptosis, migration and invasion effect and other biological characteristics, and to explore its possible mechanism. This study suggests that in epithelial eggs FKBP14 ovarian cancer may be a cancer gene, and may become a potential target for treatment. Objective to study the expression and significance of the first part of FKBP14 in epithelial ovarian carcinoma: to detect the expression of FKBP14 in ovarian cancer tissues and non cancer tissues, to explore the relationship between FKBP14 and ovarian cancer. Methods: 1. by RT-PCR method to detect the expression of 40 cases of ovarian cancer and 40 cases of non cancerous tissues in FKBP14 mRNA, the expression of.2. was used to detect whether there are differences in FKBP14 protein of SP was detected in 60 cases of different pathological types of epithelial ovarian carcinoma. The expression of the situation, in 20 cases of ovarian benign cyst tissues correlation analysis between FKBP14 and epithelial ovarian cancer. Results: epithelial ovarian carcinoma tissues, the expression level of FKBP14 protein and mRNA were significantly higher than that of control group, the difference of expression suggests that FKBP14 may be epithelial A cancer gene of ovarian cancer. Conclusion: FKBP14 may be involved in the process of occurrence and development of epithelial ovarian carcinoma. The second part of the interference suppression of FKBP14 expression on the proliferation and migration of ovarian cancer cells objective to investigate the influence of using RNA interference targeting inhibition of FKBP14 expression in ovarian cancer cell lines SKOV3 and H08910. Observation of gene silencing on the biological behavior of ovarian cancer cells: proliferation, apoptosis and invasion ability, the role of FKBP14 in the pathogenesis of ovarian cancer, as the molecular target for ovarian cancer treatment to provide the theory basis. Methods: 1. using Real-time PCR and Western blot for the detection of five ovarian cancer cell lines: A2780, OVCAR3, FKBP14 protein expression of SKOV3,3A0 and H08910, selected 2 high expression in epithelial ovarian cancer cell lines FKBP14 gene (SKOV3 and HO-8910).2. construction FK Lentiviral vector recombinant BP14 gene by gene chemical synthesis of small interfering siRNA targeting FKBP14 sequence (GACCACTTTCACTGATTAT) and non silencing sequence (CCTAAGGTTAAGTCGCCCTCG), folded into shRNA, cloned into PLKO.1 retroviral vector, and through Lipofectamine 2000 transfection HEK293 cells. After cultured for 48 hours, collect the virus reverse stability turn the target cell to the parental cells and empty vector transfected cells as compared to.3. using CCK8 assay interference group (RNAi), control group (WT group), non interference sequence (NC) proliferation of ovarian cancer cell line.4. Annexin V-FITC/PI double staining flow cytometry to detect interference group (RNAi). The blank control group (WT group), non interference sequence (NC) of ovarian cancer cell line apoptosis and cell cycle changes of.5. by Transwell assay technology, detection of interference group (RNAi), control group (WT), non interference sequence Group.6. (NC) changes in migration and invasion of ovarian cancer cell line Real-time by detecting PCR and Western blot (RNAi), interference group, blank control group (WT group), non interference sequence (NC) expression in ovarian cancer cell line related pathway proteins: E-cadherin, Twistl, MMP2, BCL-2, BAX, capspase3 PCNA,.7. SPSS 13 software was used for data analysis, measurement data with the average standard deviation analysis, independent sample t test for differences between groups. Results: 1. interference group (RNAi) and blank control group the expression of FKBP14 protein in ovarian cancer cells (WT) and non interference group (NC) compared to a decrease of 90%, WT group and NC group had no difference between the expression level of FKBP14 in.2.NC group and WT group have no statistically significant differences between cell proliferation, suggesting that shRNA'interference lentiviral transfection system no cytotoxic effect of.FKBP14 SKOV3 and H08910 stem cells in the rejection group after transfection for 48 of ovarian cancer cells Hours, 72 hours are shown to inhibit.3.FKBP14 shRNA proliferation after transfection significantly, compared with the WT group and NC group, RNAi group showed the percentage of G0/G1 phase increased, the proportion of S phase decreases the apoptosis rate of.RNAi group increased 9 times than in NC group. Cell cycle and apoptosis in NC group and WT group had no difference compared to.4. WT group and NC group, RNAi group, cell proliferation related protein PCNA, anti apoptotic protein Bcl-2 expression decreased significantly, the apoptosis marker protein Caspase3 and pro apoptotic protein Bax expression increased. In the suppression of FKBP14 expression, the ratio of Bax/Bcl-2 significantly increased the migration and invasion of.5. cells in experimental group RNAi cells, invasion, migration rates were lower than WT group and the cells of NC group.6.RNAi group of matrix metalloproteinase MMP2 and EMT inducing factor Twist expression was lower than WT group and NC group, while the epithelial marker E-cadherin was higher than that of WT group and NC group. Conclusion: 1.FKBP14 is involved in the ovarian cancer cell proliferation, apoptosis, migration Shift and invasion process, its silencing can significantly reduce cell proliferation, promote cell apoptosis and decrease the invasion and migration. It suggested that FKBP14 may be a cancer gene in the occurrence and development of ovarian cancer, we suggest FKBP14 may become a potential target for the.2. FKBP14 shRNA gene therapy for ovarian cancer GO/G1 cells through inhibition of cell cycle arrest the proliferation of FKBP14 can promote the invasion and metastasis of ovarian cancer cells by regulating the ratio of Bax/Bcl-2.3. FKBP14 inhibits apoptosis and possible mechanism of regulating epithelial mesenchymal transition (EMT) process and the effect of extracellular matrix (ECM) components.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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