【作者】 王慧；

【导师】 付艳；

【作者基本信息】 吉林大学 ， 妇产科学， 2016， 博士

【摘要】 卵巢肿瘤是妇科常见的肿瘤。卵巢癌的死亡率居妇科恶性肿瘤的首位,对妇女生命造成严重威胁。由于卵巢的组织学特点,卵巢肿瘤分类繁多,早期症状缺乏,至早期诊断异常困难。正常的卵巢组织学成分复杂,具有增殖活跃特性。目前已有多项研究支持成年哺乳动物卵巢中含有生殖干细胞(GSC)。卵巢细胞增殖活跃可能与哺乳动物的卵巢中存在的生殖干细胞(GSC)有关。肿瘤细胞的生物学行为如无限增殖、多向分化等与胚胎干细胞相似,推测肿瘤的本质有“返祖现象”。那么肿瘤细胞的发生和转移过程应当存在着与胚胎发育过程共同的调控机制。调节胚胎发育的某些因子可能也是促进肿瘤发生的影响因素。FOXC2是与早期胚胎多种组织分化、构型有关的胚胎性转录因子,与血管和淋巴管的发生有关。出生后FOXC2基因停止表达。近年发现FOXC2还与宫颈癌、内膜癌等妇科肿瘤、乳腺癌、结肠癌和黑色素瘤的发生、发展密切相关,更与肿瘤血管和淋巴管的发生有关。亦有报道指出FOXC2在肿瘤干细胞的发生、发展中起重要作用。VEGF也是新血管生成的关键分子,与肿瘤生长、侵袭性和淋巴结转移相关。VEGF通过诱导肿瘤相关的微小血管内皮细胞中Bcl–2基因的表达,促进内皮细胞迁移、分化和血管渗透,进而诱导细胞增殖和瘤内新血管形成。VEGF与内皮细胞膜上的VEGFR2结合后,通过PI3K-Akt信号通路激活Bcl–2,不仅促进癌细胞存活,还促进血管生成的效应因子趋化因子(CXCL8,IL-8)增加,同时IL-8也作为VEGF信号途径的下游效应因子有强有力的促血管生成作用,使病灶内血管加速形成,加速这些部位细胞生长。FOXC2是否在卵巢肿瘤组织的发生发展过程中发挥作用、FOXC2在类型繁多复杂的卵巢肿瘤组织表达状况如何,均未见明确报道。如果FOXC2在卵巢肿瘤组织的发生发展过程中有作用,FOXC2对哪些种类卵巢肿瘤起作用;由于IL8与VEGF在血管生成过程的有着必然联系,FOXC2是否也是通过VEGF—IL8信号途径在卵巢癌的发生、发展、新生微血管等过程中发挥作用。本实验对不同种类的卵巢肿瘤组织中的FOXC2蛋白的表达及分布进行了研究,探讨了FOXC2是否在卵巢肿瘤以及哪些种类肿瘤中发挥作用;探讨了FOXC2在肿瘤增殖与侵袭力、脉管形成上的作用和作用机制,对解明卵巢癌发生过程、预防卵巢癌淋巴转移及发现新的肿瘤诊治方法有非常重要的意义。目的:探讨FOXC2是否在卵巢肿瘤以及哪些类肿瘤中表达,FOXC2的表达对卵巢癌细胞的生物学行为有何影响以及作用机制,为解明卵巢癌发生机制、改善卵巢癌的预后及发现新的肿瘤诊治方法提供理论基础。方法:利用免疫组织化学方法对收集到的卵巢上皮性肿瘤、卵巢生殖细胞肿瘤和卵巢性索-间质细胞肿瘤3大类卵巢肿瘤组织中的FOXC2蛋白的表达及分布进行了调查,探讨FOXC2在各类卵巢肿瘤组织中的表达状况;利用FOXC2 sh RNA技术沉默人卵巢上皮性囊腺癌SKOV-3细胞的FOXC2基因表达,CCK-8法检测卵巢癌细胞的增殖能力、划痕实验检测迁移能力、细胞克隆实验检测细胞成瘤能力和侵袭实验检测肿瘤细胞的侵袭性,以及实时定量PCR和间接免疫荧光染色的方法检测分析SKOV-3细胞中FOXC2与VEGF和IL8信号传递关系。结果:1.人卵巢肿瘤组织FOXC2的表达状况(1)FOXC2在上皮性卵巢肿瘤表达上皮性卵巢肿瘤,FOXC2表达阳性率为88.13%(52/59)。浆液性肿瘤FOXC2表达阳性率低于粘液性肿瘤。在卵巢浆液性囊腺瘤,仅部分生发上皮细胞的细胞质发现低强度的FOXC2表达。在卵巢交界性浆液性囊腺瘤细胞FOXC2呈低中强度表达;CD31标记的微小血管内皮细胞质中FOXC2和VEGF强表达。在卵巢浆液性乳头状囊腺癌组织,从IC期到IIIC期,癌细胞FOXC2呈从弱到强的表达;在微小血管壁内皮细胞呈强表达。在FOXC2表达的同时,卵巢浆液性囊腺癌细胞也表达VEGF。在卵巢粘液性囊腺瘤,FOXC2表达在卵巢粘液性囊腺瘤的粘液上皮细胞质以及上皮下的间质组织细胞质;在交界性粘液性囊腺瘤,间质中血管上皮细胞细胞质和细胞核均有FOXC2表达。在粘液性囊腺癌,FOXC2主要表达在癌灶间质部癌细胞的细胞核和细胞浆及间质的微小血管壁细胞质。在卵巢勃勒纳瘤组织,没有FOXC2表达。在卵巢子宫内膜样腺癌细胞质和间质微小血管壁细胞,FOXC2呈强表达。在间质中有散在间质细胞核FOXC2呈强表达。(2)FOXC2在生殖细胞肿瘤的表达生殖细胞肿瘤FOXC2表达阳性率为40.00%(2/5)。在卵巢成熟性囊性畸胎瘤没有呈现FOXC2表达。在伴体细胞恶变的卵巢成熟性囊性畸胎瘤鳞癌病灶,间质细胞呈散在的细胞核FOXC2强表达。在III级卵巢未成熟性畸胎瘤的生发上皮有低强度的FOXC2表达。在卵巢甲状腺肿和卵巢混合性生殖细胞肿瘤(卵黄囊瘤+成熟性囊性畸胎瘤)组织没有发现FOXC2表达。(3)FOXC2在性索-间质肿瘤的表达性索-间质肿瘤,FOXC2表达阳性率为60.00%(3/5)。在高分化卵巢支持-间质细胞瘤的SERTOLI细胞没有FOXC2表达,而LEYDIG细胞核FOXC2强表达。在卵巢环状小管性索瘤(SCTAT)细胞质FOXC2强表达。在卵巢成年型颗粒细胞瘤,散在的有分裂相的细胞核FOXC2强表达。在卵巢卵泡膜纤维瘤,没有发现FOXC2表达。2.FOXC2 sh RNA对SKOV-3细胞生物学行为的影响(1)人卵巢癌细胞SKOV-3经瞬时转染FOXC2 sh RNA 24h时及以后,CCK8方法检测结果显示,FOXC2sh RNA组细胞数量较未转质粒组和sh NC组显著地减少,P﹤0.01。(2)采用划痕实验检测SKOV-3细胞迁移率,结果显示,经72h培养时,未转质粒组细胞的快速迁移使划痕消失;sh NC组划痕两侧细胞的较快生长,使划痕变得细窄,边缘不清;而FOXC2 sh RNA组划痕仍较宽,边缘清晰。(3)将转染质粒的细胞以300个/孔或以600个/孔的细胞浓度接种于6孔板培养14天后染色观察,结果显示,300个/孔的sh NC组细胞成瘤率为87.6%,600个/孔的成瘤率为90.5%均显著高于FOXC2 sh RNA组300个/孔25.3%的成瘤率,600个/孔24.7%的成瘤率。(4)将转染FOXC2 sh RNA和sh NC的细胞于transwell小室37℃5%CO2细胞培养箱中培养24 h,结果显示,转染sh NC组的侵袭细胞数目明显高于FOXC2sh RNA组。定量分析结果显示,每个视野转染sh NC组的侵袭细胞数目为(96.8±16.84),FOXC2 sh RNA组的侵袭细胞数目为(27.4±9.34),两者比较,t=8.057,P<0.001。3.si RNA靶向干扰FOXC2基因对人卵巢癌细胞VEGF和IL8影响(1)在卵巢上皮性浆液性腺癌中FOXC2主要表达在腺癌细胞胞质及周围间质血管内皮细胞中;VEGF不仅表达在间质中血管内皮细胞中,在卵巢浆液性囊腺癌癌灶中的癌细胞FOXC2高表达的区域,VEGF的表达强度同时也非常高。(2)FOXC2 sh RNA瞬时转染人卵巢上皮性囊腺癌细胞系SKOV-3细胞,FOXC2 sh RNA组FOXC2 m RNA水平较sh NC组下降了80.1%,差别显著(P<0.01);同时VEGF m RNA水平也较sh NC组下降77.8%,差别显著(P<0.01);但是,IL8 m RNA的表达水平,两组比较没有变化(P>0.05)。(3)间接免疫荧光染色显示,FOXC2 sh RNA组中FOXC2荧光强度弱于sh NC组,FOXC2 sh RNA组中的VEGF荧光强度也弱于sh NC组;FOXC2 sh RNA组FOXC2表达强度和VEGF的表达强度均较sh NC组显著减少(P<0.01)。(4)FOXC2 sh RNA和sh NC转染人卵巢癌SKOV-3细胞后,FOXC2 sh RNA组IL8表达强度与sh NC组相比没有明显变化(P>0.05)。结论:1.FOXC2在上皮性卵巢肿瘤呈高表达率。粘液性高于浆液性卵巢肿瘤。FOXC2在卵巢浆液性乳头状囊腺癌中的表达随着临床分期由IC向IIIC期明显增强。FOXC2在卵巢粘液性囊腺癌的表达强度明显高于浆液性囊腺癌。2.FOXC2在卵巢浆液性囊腺癌细胞和囊腺癌病灶新生的微血管中与VEGF同时有表达,表明VEGF与FOXC2可能存在着某种必然的联系,FOXC2可能参与卵巢肿瘤组织血管的形成。3.在人卵巢癌细胞SKOV-3有FOXC2表达。sh FOXC2靶向沉默SKOV-3细胞FOXC2基因后,FOXC2 m RNA和蛋白表达量均明显下降;可降低SKOV-3细胞的体外增殖能力、迁移能力、成瘤能力和侵袭能力。4.靶向沉默FOXC2基因使SKOV-3细胞中VEGF表达量下降,但不影响IL8的表达。FOXC2可能是VEGF的上游基因,对人卵巢癌SKOV-3细胞的作用可能是促进肿瘤细胞存活、增殖和具有侵袭力。FOXC2可能在卵巢肿瘤的发生、发展、转归过程中发挥了重要作用。更多还原

【Abstract】 Ovarian tumors are common gynecological diseases. Among these tumors, the ovarian cancer occupies the highest mortality rate of all gynecological malignancies.The ovary consists of multiple histological constituents, and malignancies could derive from these different kinds of histology, usually lacking early symptoms and challenges early diagnosis. Recent studies support the existence of GSCs in mammalian ovaries with which ovarian malignancies exhibit their resemblance,indicating an “atavism” of the nature of ovarian malignancies, leading to the corollary of shared regulation mechanism between the development of malignancies and embryos, where regulating factors during embryo development might act as crucial factors in cancer development.FOXC2 is a transcription factor that participates in multiple processes during early embryo development such as tissue differentiation, composition, and the development of blood and lymphatic vessels, whose expression generally cease after birth. In recent years, FOXC2 has been found to be associated with the development,especially the development of blood and lymphatic vessels of cervical cancer,endometrial cancer, breast cancer, colon cancer and melanoma. Other reports have described its critical functions in the development of cancer stem cells.VEGF is a critical factor in the process of neovascularization, cancer growth,invasion and metastasis. VEGF promotes the endothelial cell migration,differentiation and infiltration by inducing BCL-2 expression in the endothelial cells of microvessles, thus inducing cancer development and neovascularization. The mechanism involves the ligation of VEGF with VEGFR2 on the endothelial cell membrane, via P13K-Akt pathway, stimulates BCL-2, enhancing the survival of cancer cells while promoting the expression of CXCL8 and IL-8, which are important downstream targets during intra-lesional blood vessel formation.Up to now, no studies have focuses on the expression and function of FOXC2 in ovarian tumors of different histology. If FOXC2 is involved in the development of ovarian tumors, its detailed function and its association with VEGF-IL-8 signalingduring tumor development and neovascularization is to be clarified. In this studied,we determined the expression, distribution and function of FOXC2 in different types of ovarian tumors, and analyzed the function and mechanism of FOXC2 in tumor growth, invasion and vascularization, and deepened the understanding of ovarian tumorgenisis and metastasis.Purpose:To investigate the expression profile of FOXC2 in different types of ovarian tumors, and its effect and mechanisms on cancer biology and its association with cancer diagnosis and prognosis.Methods:The level and site of expression FOXC2 was determined by immunohistochemistry in collected pathological tissues of ovarian cancers of epithelial, germ cell and sexcord-stromal cell origin. FOXC2 expression was silenced with sh RNA in Human epithelial ovarian cystadenocarcinoma cell line SKOV-3, and its growth was determined by CCK-8 assay, migration by scratch assay, tumorgenisis by cell cloning experiment and invasion by invasion assay. Real-time PCR and indirect immunofluorescence was performed to analyze the interaction between FOXC2 and VEGF and IL-8 signaling.Results:1. The expression of FOXC2 in human ovarian cancers(1)FOXC2 expression in ovarian epithelial cancers The positive rate of FOXC2 s expression is 88.13%(52/59) in epithelial cancers.The expression in serous cancers is lower than that of mucous cancers.In epithelial ovarian cystadenocarcinomas, low levels of FOXC2 expression could be found in the cytoplasm of a portion of germinal epithelial cells. Low to intermediate expression could be detected in borderline serous cystadenoma, and strong expression of FOXC2 and VEGF detected in CD31 positive microvascular endothelial cells. An increasing expression of FOXC2 was detected from stage I C to III C serous papillary cystadenocarcinomas, with strong expression of FOXC2 in microvascular endothelial cells.The expression of FOXC2 in the ovarian mucous cystadenocarcinoma is located in the cytoplasm of mucous epithelial cells and stromal cells. In the borderline mucous cystadenomas, FOXC2 is expressed in both the cytoplasm and the nuclei of the stromal vascular epithelial cells. In mucous cystadenocarcinomas, FOXC2 is mainly expressed in the cytoplasm and the nuclei of the stromal cells and the cytoplasm of microvascular wall.(2)FOXC2 expression in ovarian germ cell cancers The positive rate of FOXC2 expression in ovarian germ cell cancers is 40%(2/5).No expression was observed in the mature cystic teratomas. In the squamous cell carcinoma lesions of mature cystic teratomas with somatic cell malignant transformation, scattered strong nuclear expression was observed in a portion of stromal cells. Low level of expression was observed in the germinal epithelial cells of Grade III ovarian immature teratoma. In ovarian thyroid cyst and ovarian mixed germ cell cancer, no expression was observed.(3)FOXC2 expression in sexcord-stromal cell cancers The expression rate is 60%(3/5). No FOXC2 expression was found in SERTOLI cells and strong nuclear expression was determined in LEYDIG cells in highly differentiated ovarian stromal cell tumors. In SCTAT, strong expression was observed in the cytoplasm. In ovarian adult granulosa cell tumors, scattered cell with karyomitosis showed stong nuclear FOXC2 expression. In fibrothecomas, no expression was found.2. The effect of FOXC2 sh RNA silencing on the biology of SKOV-3.(1)Transient transfection of FOXC2 sh RNA resulted in significantly reduced cell numbers in CCK8 assay.(2)FOXC2 silencing induced wider scratch compared with the control group and sh NC group after 72 h cell culture in the scratch assay.(3)When seeded with 300 or 600 cancer cells, the control group showed a significantly higher tumorogenisis with 87.6% in the 300 group and 90.5% in the 600 group compared with 25.3% and 24.7% in the FOXC2 silenced group.(4)Transwell assay showe significantly higher number of infiltrating cells in the sh NC group comprared with FOXC2 silencing group after 24 h cell culture.Quantification showed 96.8±16.84 in the control group vs 27.4±9.34 in the FOXC2 silencing group.3. The effect of FOXC2 silencing on human ovarian cancer VEGF and IL-8 signaling(1) silencing of FOXC2 m RNA in SKOV-3 cells resulted in 77.8% reduction of VEGF m RNA expression, while no IL-8 m RNA changes was observed.( 2) Indirect immunofluorescence determined significantly lowered VEGF signal in FOXC2 silencing group compared with the control, while no change in the signal of IL-8 was observed.Conclusion1. FOXC2 is highly expressed in the ovarian epithelial cancers, of which the mucous cancers have higher expression compared with the serous cancers. There is positive correlation between FOXC2 expression and the staging of Ovarian serous papillary cystadenocarcinoma, in which the mucous cancers have high expression than the serous cancers.2. FOXC2 is co-expressed with VEGF in the cancer cells and neovasculature of ovarian mucous cystoadenocarcinomas, suggesting an inter-connection between the two factors. FOXC2 might participate in the angiogenisis of ovarian cancers.3. FOXC2 is expressed in the SKOV-3 cells, silencing its expression has a negative effect on cancer growth, migration, invasion and tumorgenesis.4. FOXC2 silencing down regulates VEGF but not IL-8, suggesting its role as an upstream regulator.更多还原