【作者】 李磊；

【导师】 霍然；

【作者基本信息】 山东大学 ， 外科学（烧伤整形）（专业学位）， 2016， 博士

【摘要】 研究背景恶性黑色素瘤是极具侵袭性的皮肤恶性肿瘤之一,多发于欧美等发达国家。在美国,恶性黑色素瘤分别是发病率第五的男性恶性肿瘤(5%)和发病率第六的女性恶性肿瘤(4%)。对于亚洲国家而言,恶性黑色素瘤的发病率远远低于欧美国家,大部分地区发病率不足1/10万。在中国,恶性黑色素瘤并不是一种十分常见的恶性肿瘤,然而,由于人口基数大,恶性黑色素瘤的总体罹患人数较多,尤其在沿海发达城市,恶性黑色素瘤的发病率和死亡率均呈现快速上升趋势,应引起足够的重视。此外,恶性黑色素瘤是一种早期转移率极高的恶性肿瘤,预后很差,这也是该病致死率高的一个重要原因。恶性黑色素瘤病因复杂,目前已经明确的恶性黑色素瘤致病因素有遗传因素、日光暴露等环境因素以及电离辐射等因素。治疗措施方面,临床上以手术切除治疗为主,辅以免疫疗法、肿瘤靶向药物治疗等综合措施,可改善大部分恶性黑色素瘤患者的疾病进程。然而,上述治疗措施对于转移性恶性黑色素瘤疗效甚微,患者的五年生存率不足20%。因此,寻找新的诊断、治疗手段对于提高恶性黑色素瘤患者的预后具有重要意义。成纤维细胞生长因子(fibroblast growth factor, FGF)家族蛋白及其受体FGFR家族蛋白属于经典的酪氨酸激酶受体-配体信号通路转导蛋白。目前共有22种FGF和4种FGFR被发现和鉴定。FGFR家族蛋白在机体正常的血管形成、胚胎发育等过程发挥重要作用。FGFR3最早被认为是一种骨骼发育的负向调控蛋白,FGFR3敲除的小鼠呈现长骨发育过度的表型,而激活的FGFR3突变则被证实与侏儒症密切相关。近年来,过度激活的FGFR3信号被证实与骨髓瘤、宫颈癌、膀胱癌等恶性肿瘤密切相关,激活型FGFR3突变可以显著增强多种肿瘤细胞的恶性行为,因此FGFR3被认为是多种恶性肿瘤的一个潜在治疗靶点。FGF及其受体与恶性黑色素瘤的发生发展关系密切,FGFR1是决定恶性黑色素瘤原位增生、血管新生和远处转移的一个关键基因。FGFR4与恶性黑色素瘤的恶性程度正相关。考虑到FGFR家族蛋白在结构上的同源性和功能上的相似性,阐明FGFR3在恶性黑色素瘤中的表达情况、临床意义和生物学功能可能具有重要的价值。研究目的1、使用定量PCR技术明确FGFR3在恶性黑色素瘤组织和配对正常组织中的表达量。2、检测FGFR3在恶性黑色素瘤组织中的表达量与患者的临床病理特点之间的关系。3、分别在恶性黑色素瘤细胞中敲低和过表达FGFR3,验证FGFR3对细胞增殖的调控作用。4、验证FGFR3对恶性黑色素瘤细胞的体外迁移、侵袭及上皮间充质转化(epithelial-mesenchymal transitions, EMT)行为的调控作用。5、探究FGFR3对恶性黑色素瘤细胞体内增殖、迁移的调控作用。6、初步明确沉默FGFR3导致的恶性黑色素瘤细胞信号通路改变。研究方法1、收集恶性黑色素瘤组织及其癌旁组织样本42对,抽提RNA之后,采用荧光实时定量PCR技术(Quantitative Real-time PCR, qRT-PCR)检测FGFR3在恶性黑色素瘤组织和对应癌旁组织的表达量。2、免疫组化芯片技术检测FGFR3在恶性黑色素瘤组织中的表达量,根据染色强度和阳性细胞数进行评分。染色强度由低到高分为A、B、C、D四个等级,阳性细胞数分为A(<10%)、B(10%-25%)、C(25%-50%)、D(>50%)四个等级。采用染色强度的评分乘以阳性细胞数评分代表每个样本最终的评分,0-3分代表FGFR3低表达,4-9代表高表达。3、采用慢病毒转染技术获得稳定敲低FGFR3的细胞株A375/LV-shFGFR3和对照细胞株A375/LV-control。瞬时转染技术获得高表达FGFR3的细胞株A375/pcDNA3.0-FGFR3和对照细胞株A375/pcDNA3.0,采用qPCR技术分别验证mRNA水平的敲低效率和过表达效率。4、采用CCK-8技术检测FGFR3对恶性黑色素瘤细胞增殖的调控作用。使用适量胰酶消化细胞后,用培养液重悬,反复吹打均匀,将其浓度调整为1000细胞/100微升。各组细胞分别取100ul细胞悬液置于96孔板中,每天相同时刻加入10ul CCK-8,4h后使用酶标仪检测吸光度值,连续检测6天。5、采用流式细胞术检测FGFR3对恶性黑色素瘤细胞凋亡率的影响。A375/LV-shFGFR3和A375/LV-control细胞经AnnexinV和PI染色后,经流式细胞仪检测其凋亡率差异。6、采用transwell小室和matrigel invasion chambers分别检测FGFR3对恶性黑色素瘤细胞迁移、侵袭的调控作用。7、A375/LV-shFGFR3和A375/LV-control细胞分别注射入4周龄裸鼠右侧腋下,每周检测肿瘤体积变化,5周后将裸鼠处死,对比两组皮下移植瘤的大小和重量。两组细胞分别经尾静脉注射入裸鼠体内,两个月后对比两组裸鼠肺部转移结节的数目。8、采用Western blot技术检测A375/LV-shFGFR33和A375/LV-control细胞各自的ERK、AKT、EGFR等关键信号通路蛋白及其磷酸化蛋白的表达量。研究结果1、FGFR3在恶性黑色素瘤组织中的表达量显著高于癌旁组织。FGFR3在恶性黑色素瘤组织中的表达量与Breslow厚度和淋巴结转移相关。2、慢病毒干扰FGFR3的表达之后,A375细胞增殖活力降低,质粒过表达FGFR3之后,A375细胞的增殖活力升高。3、沉默FGFR3显著增加恶性黑色素瘤细胞的凋亡率,过表达FGFR3降低其凋亡率。4、慢病毒干扰FGFR3的表达之后,恶性黑色素瘤A375细胞的迁移、侵袭能力降低,而FGFR3表达质粒可增强恶性黑色素瘤细胞的迁移和侵袭能力。5、沉默FGFR3可以显著降低恶性黑色素瘤细胞的体内增殖、迁移能力。6、沉默FGFR3曾加上皮性标志物E-cadherin、Laminin的表达量,降低间充质标志物N-cadherin、vimentin等的表达量。7、沉默FGFR3对ERK、AKT、EGFR等信号通路蛋白的表达量无明显影响,可显著下调ERK、AKT、EGFR等信号通路蛋白的磷酸化水平。研究结论FGFR3在恶性黑色素瘤组织中存在过表达；与恶性黑色素瘤的Breslow厚度和淋巴结转移正相关。FGFR3通过调控p-ERK、p-AKT和p-EGFR的水平调控黑色素瘤A375细胞的恶性生物学行为。更多还原

【Abstract】 BackgroundMalignant melanoma is one of the most invasive tumors worldwide with a higher incidence in developed countries in Europe and America. According to the statistical results by World Health Organization (WHO), the standardized incidence rate of melanoma in developed countries of Europe and America is more than 9/100 000 while this rate is less than 1/100 000 in less developed countries. In United States, malignant melanoma is the fifth most common tumors of male and the sixth most common tumors of female. As to Asian countries, the incidence of malignant melanoma is far less than the United States with an incidence rate less than 1/100000 in most areas. As to china, the incidence rate of malignant melanoma exhibits a continuous upward trend in the last decade, in addition, the total number of melanoma patients remains worrisome due to a huge population base. The poor prognosis of melanoma patients is mainly caused by early metastasis, additionally, recent studies about melanoma have made much progression and revealed that the risk factors of malignant melanoma including genetic risk, immune, and environmental factors and multiple therapeutic measures. However, the prognosis of metastatic melanoma patients remains poor with a 5-year survival of 15%. Thus, investigating for novel diagnostic and therapeutic targets for malignant melanoma is of great value.Fibroblast growth factor (FGF) and its receptor FGFR family consist a relatively conservative signaling pathway.22 kinds of FGFs and 4 kinds of FGFRs have been identified to now. FGFR3 was initially identified as a modulator of angiogenesis and embryonic development. FGFR3 knockout mice exhibited overgrowth of the long bones, while activated FGFR3 mutations were confirmed to be associated with dwarfism, indicating the negative role of FGFR3 in bone development. In recent years, overactivation of FGFR signaling had been identified to play critical rules in myeloma, cervical cancer, bladder cancer and other malignancies. Activated FGFR3 mutations could significantly enhance the malignant behaviors of tumor cells. Thus, FGFR3 had been considered as a potential therapeutic target for a variety of malignant tumors. Recently, the relationship between FGFRs and malignant melanoma had drawn much attention. FGFR1 could regulate the proliferation and angiogenesis ability of melanoma cells and silencing FGFR1 could significantly inhibit proliferation and poorly differentiated of melanoma cells. These studies indicated FGFR1 might be an oncogene of malignant melanoma. Considering the homology of FGFR family proteins, we wanted to explore the clinical significance of FGFR3 expression in melanoma tissues and the biological function of FGFR3 in melanoma cells.Objective1. The detection of FGFR3 expression in malignant melanoma tissues and non-tumor tissues.2. Evaluation of the relationship between FGFR3 expression and clinicopathological parameters of melanoma patients.3. Knocking down or overexpressing FGFR3 in melanoma cells to evaluate the role of FGFR3 on cell proliferation.4. Evaluating the role of FGFR3 on cell migration, invasion and EMT behaviors in vitro.5. Evaluating the role of FGFR3 on cell proliferation and migration in vivo.6. Detecting the change of signaling pathways after FGFR3 down-regulation.Methods1.42 paired melanoma samples were collected and the expression levels of FGFR3 in melanoma tissues and corresponding non-tumor tissues were evaluated by Quantitative Real-time PCR (qRT-PCR) and western blot respectively.2. The relationship between FGFR3 expression and the clinicopathological parameters of melanoma patients was evaluated by immunohistochemical staining, which was scored according to staining intensity and number of positive cells. Staining intensity was scored as 0-3 representing negative(-), weak(+), moderate(++), and strong (+++) staining, respectively. The stained cells were counted and scored as 0 (<10%),1 (10%-25%),2 (25%-50%),3(>50%). The final score was determined by two independent researchers. After multiplying score of staining intensity and stained cell numbers. The final score range was 0-9, and scores of 0-3 represent low expression level of LIFR and 4-9 represent high level.3. Establishment of cell lines expressing shFGFR3 or shNC by corresponding lentivirus product and cell lines expressing pcDNA3.0-FGFR3 or pcDNA3.0 by corresponding plasmids. The down-regulation or up-regulation efficiency was confirmed by qRT-PCR and western blot, respectively.4. The role of FGFR3 on cell proliferation was evaluated by CCK-8 assay. Cells at logarithmic growth phase were trypsinized, resuspended and plated into 96 well plate at a density of 1.0×104/ml with a total volume of 100ul. lOul CCK-8 was added into each plate at the same time of each day for 6 days.5. The role of FGFR3 on cell apoptosis was examined by flow cytometry. After stained by Annexin V and PI, the apoptosis rate of A375/LV-shFGFR and A375/LV-shNC cells were detected by flow cytometer respectively.6. The role of FGFR3 on cell migration and invasion was evaluated by transwell and matrigel invasion chambers according to the manufacturer’s protocol.7. A375/LV-shFGFR3 and A375/LV-shNC cells were subcutaneously injected into the right flank of 4-week-old male nude mice (5 mice per group). The tumor volume was examined every week. The subcutaneous tumors were removed for evaluating the volume and weight. As to lung metastasis assay, A375/LV-shFGFR3 and A375/LV-shNC cells were injected into tail vein of nude mice and the metastatic nodes were calculated respectively.8. Western blot assay was employed to examine the expression levels of ERK, AKT, EGFR and the corresponding phosphorylation proteins after FGFR3 down-regulation.Results1. The expression level of FGFR3 was significantly higher in melanoma tissues compared with adjacent non-tumor tissues. FGFR3 expression in malignant melanoma tissues was correlated with Breslow thickness and lymph node metastasis (p<0.05).2. Silencing FGFR3 inhibited the proliferation ability of melanoma cells while FGFR3 restoration promoted the proliferation ability.3. Silencing FGFR3 promoted the apoptosis rate of melanoma cells while FGFR3 restoration inhibited apoptosis.4. Silencing FGFR3 inhibited the migration and invasion ability of melanoma cells in vitro while FGFR3 restoration promoted the migration and invasion ability in vitro.5. Silencing FGFR3 inhibited the proliferation and migration ability of melanoma cells in vivo.6. The levels of epithelial marker E-cadherin was up-regulated while the levels of mesenchymal markers N-cadherin and vimentin were down-regulated after silencing FGFR3.7. The expression level of ERK, AKT and EGFR didn’t show much change after FGFR3 down-regulation while the phosphorylation levels of these proteins were significantly decreased.ConclusionsFGFR3 was overexpressed in malignant melanoma tissues and correlated with Breslow thickness and lymph node metastasis. FGFR3 regulated malignant melanoma cell proliferation, migration and EMT behaviors via influencing the phosphorylation level of ERK, AKT and EGFR.更多还原