【作者】 戚红；

【导师】 孙保存；

【作者基本信息】 天津医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 卵巢癌(Ovarian Carcinoma, OC)是女性常见的恶性肿瘤之一,其发病率居宫4颈癌和宫体癌之后,在女性生殖道恶性肿瘤中位于第三位。70%患者在就诊时候肿瘤就已为晚期,且多发生转移,成为女性恶性肿瘤中威胁最大的疾病。血管生成拟态(Vasculogenic Mimicry,VM)是由肿瘤细胞自身变形模仿内皮细胞组成的微循环管道,内壁无内皮细胞衬覆,为肿瘤组织提供血液循环,从而保持肿瘤组织内环境的稳定从而防止肿瘤内部坏死的发生。VM与肿瘤的生长、侵袭、转移以及病人预后密切相关,存在VM的患者生存期短,预后差。上皮间充质转变(Epithelial to Mesenchymal Transition, EMT)是指上皮源性细胞在特定的生理和病理条件下向间质源性细胞发生转化的一种现象,是VM形成的关键步骤。Wnt5a是EMT发生的重要信号通路之一。Wnt5a信号通路既能活化经典的Wnt/β-catenin通路又能活化非经典Wnt通路,并且二者之间相互作用,可能导致Wnt5a在不同肿瘤中生物学作用并不一致。因此,研究Wnt5a在卵巢癌VM中的作用及其分子机制有助于丰富和完善肿瘤VM的理论研究。目的本研究旨在探讨Wnt5a在卵巢癌血管生成拟态形成中的作用及其可能分子机制。探讨Wnt5a在卵巢癌细胞EMT发生和VM形成中的分子机制有助于丰富和完善VM理论,对肿瘤血管生成的研究具有重要意义,同时为临床上抗肿瘤治疗提供了新的思路。方法1.首先对前期实验收集来自天津医科大学附属肿瘤医院的79例卵巢癌患者手术切除且随访资料完整的临床肿瘤组织标本进行Wnt5a、PKCa和CD44的免疫组织化学染色。并复习前期实验的CD31/PAS双染结果,重点观察和分析Wnt5a表达的临床特征以及Wnt5a与VM、Wnt5a与PKCa、Wnt5a与CD44之间的关系。2.根据卵巢癌细胞系内源性Wnt5a基础表达量的水平,分别构建稳定上调和稳定下调Wnt5a表达的稳转细胞系。3.构建体外卵巢癌细胞三维培养模型,观察Wnt5a对卵巢癌细胞体外管道化形成能力的影响。4.构建体外卵巢癌细胞侵袭和迁移模型,观察Wnt5a对卵巢癌细胞体外侵袭和迁移能力的影响。5.免疫荧光染色观察和Western Blotting实验Wnt5a对卵巢癌细胞体外EMT相关蛋白的影响。6. Western Blotting实验分析Wnt5a对PKCa和EMT相关蛋白表达水平的改变情况。结果1. Wnt5a表达与卵巢癌转移和VM密切相关,是卵巢癌患者一个独立的预后指标。应用SPSS统计软件分析Wnt5a和主要临床病理参数之间的关系,Wnt5a和卵巢癌的转移/复发密切相关(P=0.008),与患者年龄、肿瘤大小、组织学分型和分级、腹水等临床病理参数无关(P>0.05)。Wnt5a阳性细.中的VM阳性率(15/25,60%)明显高于Wnt5a阴性组中的VM阳性率(8/54,14.8%),且差异具有统计学意义(P=0.000)。生存分析显示,Wnt5a阴性表达患者生存时间长于Wnt5a阳性表达患者,具有统计学意义(P=0.00)。2.卵巢癌组织中Wnt5a的表达与PKCa的表达相关。79例卵巢癌患者中有35例阳性,阳性率44.3%(35/79)。Wnt5a阳性组的PKCa表达率(21/25,84%)明显高于Wnt5a阴性组(14/54,25.9%),且差异具有统计学意义(P=0.000)。3.卵巢癌组织中Wnt5a的表达与CD44的表达相关。79例卵巢癌患者中有29例阳性,阳性率36.7%(29/79)。Wnt5a阳性组的CD44表达率(21/25,84%)明显高于Wnt5a阴性组(8/54,14.8%),且差异具有统计学意义(P=0.000)。4. Wnt5a在体外能促进卵巢癌细胞系的管道化能力。高分化的卵巢癌细胞系SKOV3形成管道能力较弱,只在边缘部分形成少量或不完整的管状结构,而低分化的卵巢癌细胞系OVCAR3形成管道能力较强,形成管状结构较多且完整,管壁较厚,有多层细胞组成。上调Wnt5a后,SKOV3细胞系形成管道能力明显增加；下调Wnt5a后,OVCAR3细胞系形成管道化能力明显受到抑制,不能形成完整的管道,差异具有统计学意义。5. Wnt5a能促进卵巢癌细胞系EMT发生。SKOV3细胞系上调Wnt5a后,免疫荧光显示其间充质标记分子Vimentin表达明显升高,而上皮标记分子E-cadherin表达明显降低,下调OVCAR3细胞系的Wnt5a表达后,免疫荧光显示其间充质标记分子Vimentin表达明显降低,而上皮标记分子E-cadherin表达明显升高,差异具有统计学意义。WB相应的检测EMT表征性蛋白的改变情况与免疫荧光结果一致。6. Wnt5a在体外能促进卵巢癌细胞系的侵袭和迁移能力。划痕实验结果显示,SKOV3转染上调Wnt5a贡粒后,伤口愈合趋势明显升高；Ovcar3转染下调Wnt5a质粒后,伤口愈合趋势明显降低,差异具有统计学意义。Transwell实验结果显示,OVCAR3细胞系经过下调Wnt5a之后,穿透能力明显降低;SKOV3细胞系经过上调Wnt5a处理之后,穿透能力明显高,差异具有统计学意义。7. Wnt5a通过PKCa言号通路促进卵巢癌细胞发生EMT,形成VM,PI3K和Snail、MMP-2等调控因子发挥重要作用。WB检测显示,将OVCAR3细胞系下调Wnt5a后PKCa表达明显降低,而将SKOV3细胞系上调Wnt5a后PKCa的表达明显升高,差异具有统计学意义。加入PKCa抑制剂后,SKOV3-pWnt5a细胞系的迁移能力明显下降,提示PKCa在Wnt5a通路中起重要作用,差异具有统计学意义。OVCAR3细胞系下调Wnt5a之后,PI3K和Snail、MMP-2表达水平显著降低,加入PKCa激活剂(PMA)之后,二者的表达水平明显升高；SKOV3细胞系上调Wnt5a之后,PI3K和Snail、MMP-2表达水平显著升高,但加入PKCa抑制剂之后,二者的表达水平则明显降低,但β-catenin的表达水平无明显差异。结论1. Wnt5a表达与卵巢癌患者的转移密切相关：Wnt5a高表达的卵巢癌患者预后差刁Wnt5a氐表达的卵巢癌患者,Wnt5a可以作为卵巢癌患者预后的一个指标。2. Wnt5a能通过促进卵巢癌细胞系发生EMT,进而促进卵巢癌细胞VM的形成,并能促进其迁移和侵袭能力,在卵巢癌演进中发挥重要作用。3. PKCa在Wnt5a信号通路中起重要作用：Wnt5a主要通过PKCa信号通路而不是β-catenin信号通路来促进卵巢癌细胞EMT发生,Snail、PI3K、MMP-2是重要调节因子4.卵巢癌干细胞特性在VM形成过程中起关键作用,Wnt5a能促进卵巢癌干细胞表型的表达,为VM形成提供生物学基础。更多还原

【Abstract】 Ovarian cancer (OC) is one of the most common causes of cancer-related death in women. Metastasis had occurred in70%of patients when they were diagnosed. The mortality rate of ovarian cancer patients has not improved although diagnosis and treatments have advanced significantly in recent years. Therefore, advances in ovarian cancer treatment must be supported by a deep understanding of its biological features. Vasculogenic Mimicry (VM) describes the ability of aggressive cancer cells to form vasculogenic-like networks in vivo. These networks lack endothelial cells or fibroblasts, and this lack is concomitant with the expression of vascular cell-associated molecules. The prognosis of patients with VM is significantly worse than that of patients without VM. EMT (Epithelial Mesenchymal Transition) is pivotal in malignant tumor progression and VM formation. However, the molecular mechanism of VM remains unclear. Wnt5a is an important noncanonical Wnt pathway that plays key role in VM and EMT. Wnt5a may active the canonical Wnt/β-catenin pathway and noncanonical Wnt pathway. So Wnt5a plays different roles in tumor. Investigating the role and mechanism of Wnt5a in VM and EMT will contribute the theory of microcirculation of tumor.ObjectiveThe study aims to investigate the function of Wnt5a in VM and EMT and analyze its mechanism in ovarian cancer. Exploring the mechanism of Wnt5a in VM and EMT will enhance our understanding of cancer biology and may identify new therapeutic approaches.Methods1. Immunohistochemistry was used to evaluate the expression of Wnt5a, PKCa and CD44in79ovarian cancer cases through Tianjin Cancer Hospital. SPSS was used to analyze the relationship between Wnt5a and clinicopathologic ovarian cancer characteristics. And we analyze the relationship between Wnt5a and VM, Wnt5a and PKCa, Wnt5a and CD44.2. We established a stablely transfected cell lines overexpression or knock-down Wnt5a expression according to the basical expression of ovarian cancer cell lines.3. A well-established model of Three-dimensional Matrigel culture in vitro was used to test the change of vasculogenic mimicry formation affer overexpression or knock-down the Wnt5a expression of ovarian cancer cells.4. The effect of Wnt5a on the motility and invasion of ovarian cancer cells in vitro was investigated. Cell migration was evaluated by wound healing assay, also known as the "scratch" assay, and cell invasion was examined by transwell assay.5. Immunofluorescence microscopy staining was performed to confirm the effect of Wnt5a on EMT assoctiated proteins.6. The expression levels of Wnt5a, PKCa, Snail, P13K, MMP-2,β-catenin were assessed using Western Blotting.Results1. Wnt5a expression was significantly associated with VM in ovarian cancer and its clinicopathologic characteristics.Of the79ovarian cancer cases in this study,25(31.6%) exhibited positive Wnt5a expression and54(68.4%) exhibited negative Wnt5a expression. The result showed that Wnt5a expression was significant relationship with cancer metastasis (P<0.05). The expression of Wnt5a was shown to be significantly correlated with VM (P<0.01). The survival time of79ovarian cancer patients was analyzed based on Wnt5a expression, and Wnt5a-positive patients were revealed to survive longer than Wnt5a-negative patients.2. Wnt5a expression was significantly associated with PKCa in ovarian cancer. Of the79ovarian cancer cases,35(44.3%) exhibited positive PKCa expression and44(55.6%) exhibited negative expression. The Wnt5a staining was found to be significantly correlated with PKCa (P<0.01).3. CD44expression was significantly associated with VM in ovarian cancer. Of the79ovarian cancer cases,29(36.7%) exhibited positive CD44expression and50(63.3%) exhibited negative expression. The Wnt5a staining was found to be significantly correlated with CD44(P<0.00).4. Wnt5a enhanced the vasculogenic capacity of ovarian cancer cells in vitro. Ovcar3, a poorly differentiated ovarian cancer line, displayed higher vasculogenic capacity than Skov3, a well-differentiated cell line. However, Ovcar3transfected with Wnt5a shRNA displayed low vasculogenic capacity, and Skov3transfected with Wnt5a cDNA exhibited high vasculogenic capacity.5. Wnt5a enhanced EMT in ovarian cancer cells in vitro.Immunofluorescence and western blot were performed to investigate the changes in the expressions of these two proteins after in vitro Wnt5a transfection. The result revealed that vimentin expression increased and E-cadherin expression decreased in Skov3after Wnt5a upregulation. By contrast, vimentin expression decreased and E-cadherin expression increased in Ovcar3after shWnt5a transfection.6. Wnt5a increased the motility and invasion of ovarian cancer cells in vitro.The results showed that Wnt5a overexpression enhanced cell migration and invasion in Skov3cells with pWnt5a (P<0.05). These findings corresponded with the weakened motility and invasion in Ovcar3cells with shWnt5a as a result of Wnt5a downregulation (P<0.05).7. Wnt5a promoted ovarian cancer via the PKCa pathway in vitro.The effect of Wnt5a expression on PKCa was investigated via western blot analysis. The results showed that changes in Wnt5a expression impaired changes in PKCa expression. The addition of PKCa inhibitor (1μM) to Skov3-pWnt5a cells significantly reduced cell motility. This result indicated that PKCa is critical to the effect of Wnt5a on ovarian cancer cells in vitro. Finally, Wnt5a and PKCa mechanisms were determined by investigating the changes in the two ovarian cancer cell lines at the protein level and analyzing the proteins associated with VM, EMT, and ECM remodeling. The findings showed that PI3K, Snail, and MMP2expression increased with Wnt5a upregulation. However, β-catenin was not affected.Conclusions1. Wnt5a expression was correlated with metastasis in ovarian cancer and was associated with poor prognosis.2. Wnt5a promoted EMT and VM in ovarian cancer, via PKCa pathway but not the (3-catenin pathway. Snail, PI3K, MMP-2play important roles in this way. 3. Wnt5a increased the metastasis and invasion of ovarian cancer cells in vitro.4. Wn5a was correlated with CD44expression in ovarian cancer, indicating that Wnt5a may be involved in cancer stem like phenotype.更多还原