【作者】 黄莹雪；

【导师】 孙建方；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2014， 博士

【摘要】 皮肤恶性黑素瘤(cutaneous malignant melanoma, CMM)是皮肤科致死率最高的恶性肿瘤,具有进展快、易转移、放化疗不敏感等特点。目前黑素瘤发生的分子机制尚不完全清楚。蛋白酶抑制剂是一种多功能蛋白质肽分子,因其在多种肿瘤中表达异常,并呈现促进和抑制的双向调节作用,成为肿瘤研究的热点之一。本研究首次探讨两种蛋白酶抑制剂卵固蛋白2(Ovostatin2, OVOS2)和组织型基质金属蛋白酶抑制剂4(tissue inhibitor of metallopmteinase-4, TIMP-4)在黑素瘤发生发展中的作用。一、蛋白酶抑制剂OVOS2与皮肤恶性黑素瘤发病机制的相关性研究1.蛋白酶抑制剂OVOS2在恶性黑素瘤组织和细胞中的表达及意义我们前期采用一款全新的全基因组表达谱芯片,发现OVOS2在中国人肢端黑素瘤中呈明显的高表达,为验证基因芯片结果,探讨蛋白酶抑制剂OVOS2表达是否与皮肤黑素瘤的发生发展过程相关,我们通过real-time PCR、Westernblot分析,发现CMM组织中OVOS2mRNA和蛋白水平较瘤旁组织明显上调；与正常人黑素细胞相比,黑素瘤细胞株中OVOS2mRNA和蛋白表达也明显上调,以在A375细胞中上调最为明显。为进一步探讨OVOS2表达与黑素瘤发生发展的相关性,我们进一步通过免疫组化方法检测OVOS2在114例CMM及良性痣组织中的表达,并分析其与临床病理特征及预后指标的关系。结果再次证实OVOS2在CMM中表达增加,其表达水平与黑素瘤恶性程度指标如Breslow厚度、Clark分级、临床分期、KI指数及VEGF表达呈正相关。可见,OVOS2在恶性黑素瘤细胞及组织中均呈现异常高表达,其表达水平与CMM的恶性程度密切相关。2.VshRNA-OVOS2重组质粒及稳定转染细胞株的构建前文实验结果表明A375细胞株中OVOS2上调最为明显,我们将A375细胞作为研究对象,首先设计4个OVOS2-shRNA片段,并载入pGMLV-SB1RNAi,构建慢病毒干扰载体(VshRNA-OVOS2),包装病毒并感染A375细胞,采用Real-time RT-PCR和Western blot筛选出沉默效果最好的靶细胞,并命名为A375/OVOS2-shRNA,同时将阴性对照慢病毒载体感染A375细胞作为对照组(negative control, NC),命名为A375/NC-shRNA。最后经细胞免疫化学法证实,A375/OVOS2-shRNA中OVOS2表达明显受抑制,所获得的稳定转染细胞株,用于后续实验。3.蛋白酶抑制剂OVOS2对A375细胞生物学行为的影响及机制研究为探讨OVOS2对人黑素瘤细胞株A375生物学行为的影响及其可能机制,我们采用直接细胞计数法、MTT法及克隆形成实验检测细胞增殖能力,流式细胞技术检测细胞凋亡及细胞周期变化,细胞划痕实验及Transwell小室实验检测细胞运动及侵袭能力,裸鼠皮下成瘤实验评估细胞体内致瘤能力,观察未处理A375细胞、A375/OVOS2-shRNA及A375/NC-shRNA三组细胞的体内外生物学行为改变；进一步通过Western blot方法检测细胞中p-FAK、p-AKT、p-ERK、 MMP-2、E-cadherin、N-cadherin和β-catenin以及细胞周期相关蛋白表达情况,分析OVOS2影响黑素瘤细胞生物学行为的可能机制。结果表明,干扰OVOS2表达能够有效抑制黑素瘤细胞A375的体内外增殖活性、导致细胞周期发生G2/M阻滞,但对细胞凋亡促进作用不显著。OVOS2可能通过过度激活FAK/MAPK/ERK和FAK/PI3K/AKT信号通路,对细胞周期和细胞增殖进行调控。沉默OVOS2表达后,细胞的迁移和运动能力显著下降,细胞中上皮表型蛋白E-cadherin,β-catenin表达增加,而间质表型蛋白N-cadherin表达降低。OVOS2可能参与黑素瘤细胞的上皮间质转化,影响肿瘤细胞的迁移和运动能力。二、蛋白酶抑制剂TIMP-4在恶性黑素瘤组织和细胞中的表达及意义通过Western blot分析,我们发现TIMP-4蛋白水平在CMM组织中较瘤旁组织和良性痣组织明显上调；黑素瘤细胞株中TIMP-4表达较正常人黑素细胞也明显上调。为进一步探讨TIMP-4表达与黑素瘤发生侵袭的相关性,我们通过免疫组化方法检测TIMP-4在94例CMM及良性痣组织中的表达,分析其与CMM临床病理特征和预后指标的相关性。结果进一步证实TIMP-4在CMM中表达升高,其表达水平与黑素瘤恶性程度及MMP-2和VEGF表达相关。TIMP-4表达可能参与CMM的恶性进程,尤其与肿瘤转移倾向及血管生成关系密切。结论在恶性黑素瘤细胞及组织中,蛋白酶抑制剂OVOS2和TIMP-4表达均明显上调,且二者的高表达均与黑素瘤的恶性程度相关。体内外实验表明阻断OVOS2表达可明显降低黑素瘤细胞A375的增殖活性及迁移运动能力,影响细胞周期进程,OVOS2对肿瘤的正向调控作用可能与其过度激活FAK/MAPK/ERK和FAK/PI3K/AKT信号通路,促进上皮间质转化过程有关。可见,蛋白酶抑制剂OVOS2和TIMP-4与恶性黑素瘤的发生发展关系密切,OVOS2可能在促进黑素瘤细胞的增殖和迁移运动过程中起到重要作用,而TIMP-4表达上调则反映肿瘤转移倾向及血管生成潜能。这些研究结果为恶性黑素瘤的诊断和治疗提供了新的研究方向和思路,充实了蛋白酶抑制剂与黑素瘤发生的相关理论,为进一步研究它们的确切作用及分子机制提供了大量的理论基础和实验依据。更多还原

【Abstract】 Cutaneous malignant melanoma (CMM) is characterized by aggressive invasion, early metastasis, and resistance to chemotherapy or radiotherapy, accounting for the highest lethality of all dermatological cancers. The molecular mechanisms leading to melanoma are still poorly understood. Protease inhibitors are multifunctional proteins, which display an abnormal expression in several kinds of tumors. Recent data indicate that protease inhibitors have both inhibitory effect and stimulatory effect on tumorigenesis. Due to its dual regulation on tumor progression, protease inhibitors become one of the top areas of cancer research. In the current study, functions of two protease inhibitors OVOS2and TIMP-4in pathogenesis and progression of melanoma were investigated for the first time.Part1:Involvement of proteinase inhibitor Ovostatin2(OVOS2) in pathogenesis of cutaneous malignant melanoma1) Expression of Ovostatin2(OVOS2) in human cutaneous malignant melanoma tissues and cellsWe previously found the expression of OVOS2gene was apparently higher in acral-melanoma tissues of Chinese patients through an advanced whole-genome oligonucleotide microarray. To confirm results of the preliminary microarray analysis, and to see if expression of OVOS2is associated with the development of CMM, real-time PCR and Western blot analysis were used to detect mRNA and protein expression of OVOS2. The results revealed that OVOS2was overexpressed in mRNA and protein level in CMM compared with paired normal skin and in melanoma cells compared with primary melanocytes. Of four kinds of melanoma cells, A375presented the highest expression level. To investigate the relationship of OVOS2expression with the progression of CMM, immunohistochemical staining was further performed on114tissue samples taken from CMM and benign nevi, followed with clinicopathological significance analysis. We confirmed OVOS2was significantly up-regulated in CMM again, and found the expression of which positively correlated with the known prognostic variables including clinical stage, Clark level and Breslow depth, and significantly associated with the status of ulcer, positivity of Ki-67and expression of VEGF in primary melanoma. These results proved the over-expression of OVOS2in melanoma tissues and cells, and suggested a relationship of OVOS2expression and aggressive behaviours of melanoma.2) Inhibition of the expression of OVOS2by VshRNAWe chosed A375as target cells as they presented the highest expression level of OVOS2. Four shRNA fragment for OVOS2were synthetized and subcloned into pGMLV-SB1RNAi lentiviral vectors respectively(VshRNA-OVOS2). Lentivirus package was conducted in293T cells, followed by transfection with the four OVOS2-shRNA into A375cells. The optimum shRNA fragment against OVOS2was determined by Real-time PCR and Western blot and the corresponding stably transfected A375cells (A375/OVOS2-shRNA) were chosen for further study. A mock lentiviral vector was transfected into A375cells as a negative control (A375/NC-shRNA). Immunocytochemistry confirmed OVOS2expression was obviously inhibited in OVOS2-shRNA transfected A375cells compared with negative control-shRNA transfected A375cells and untreated A375cells. The stably transfectant clones were used for further study.3) Effect of OVOS2on biological behaviours of A375cell and mechanism research To evaluate the effect of inhibition of OVOS2on biological behaviours of A375cell and investige the possible involved mechanisms, biological behaviours of untreated A375cell, A375/NC-shRNA and A375/OVOS2-shRNA were evaluated and compared with each other. Cell proliferation ability was observed by cell number counting, MTT assay and Colony formation assay; Cell cycle and apoptosis were detected by Flow cytometer; Cell migration and invasive ability was tested by wound-healing and Transwell study. We also observe the subcutaneous transplanted tumor growth in nude mice to study the proliferation capability in vivo. Western blot was used to detect expression of p-FAK, p-AKT, p-ERK, MMP-2, E-cadherin, N-cadherin, β-catenin and cell cycle regulatory proteins to investigate the possible mechanisms of OVOS2involving in biological behaviours of melanoma cells. These in vivo and in vitro results manifested that inhibition of OVOS2had significantly suppressive effects on the growth of A375, with a G2/M phase arrest in cell cycle process, but affected cell apoptosis rate weakly, indicating high expression of OVOS2can promote cell cycle process and enhance proliferation of melanoma cells. The involved mechanisms may be associated with its over-activation of FAK/MAPK/ERK and FAK/PI3K/AKT signals. Furthermore, down-regulation of OVOS2can reduce motility and migration capability of melanoma cells significantly, accompanied with up-regulated epithelial phenotype E-cadherin and β-catenin and down-regulated mesenchymal phenotype N-cadherin expression, suggesting an important role of OVOS2in process of cell motality and migration, and the underlying mechanisms may related to the epithelial-mesenchymal transition of melanoma cells. Part2:Expression of tissue inhibitor of metalloproteinase-4(TIMP-4) in CMM tissue and cellsWestern blot revealed that TIMP-4was overexpressed in CMM tissues compared with paired normal skin and benign nevi and in melanoma cells compared with primary melanocytes. To investigate the relationship of OVOS2expression with the development and progression of CMM, immunohistochemical staining was further performed on94cases tissue samples taken from CMM and benign nevi, followed with clinicopathological significance analysis. We confirmed TIMP-4was significantly up-regulated in CMM again, and found the expression of which was positively correlated with the progression of CMM. Furthermore, it was associated with the expressions of VEGF and MMP-2. These results suggest TIMP-4protein may be closely associated with tumor initiation and progression, especially with the process of metastasis and angiogenesis of CMM.ConclusionsProteinase inhibitors OVOS2and TIMP-4were significantly overexpressed in melanoma tissues and cells, and the expression of which was positively correlated with the aggressive progression of CMM. The in vivo and in vitro results manifested that inhibition of OVOS2had significantly suppressive effects on the proliferation, motility and migration capability of A375, and can blocked cell cycle process, suggesting a promotive role of OVOS2in pathogenesis of melanoma. The involved mechanisms may be associated with the over-activation of FAK/MAPK/ERK and FAK/PI3K/AKT signals and the enhancement of epithelial-mesenchymal transition caused by over-expressed OVOS2. These results suggest proteinase inhibitors OVOS2and TIMP-4may play an important role in development and progression of CMM, as OVOS2mainly effects cell proliferation and migration, while overexpression of TIMP-4may be associated with the potential of metastasis and angiogenesis in CMM. The current study enriched the concept that protease inhibitors are multifunctional with key regulation on tumor progression, which represents new insights and areas for diagnostic and therapeutic research of melanoma, and provided valuable theoretical basis and experimental data for further investigation on the precise function and molecular mechanisms of these two proteinase inhibitors.更多还原