Notch信号相关受体/配体在皮肤恶性黑素瘤中的表达

目的 检测Notch信号通路中Notch1、Notch4、Jagged1以及Dll4在皮肤恶性黑素瘤组织中的表达,初步探讨Notch信号通路在黑素瘤发病机制中的作用.方法 免疫组化SP法检测40例恶性黑素瘤及15例色素痣石蜡标本中Noteh1、Notch4、Jagged1以及Dll4的表达模式和表达强度.采用SPSS21.0软件进行卡方检验及Spearman秩相关分析.结果 在40例黑素瘤组织(31例阳性表达)及15例色素痣组织(3例阳性表达)Notch1的表达率差异有统计学意义(x2=15.281,P=0.000),原位(18例)及侵袭性黑素瘤(22例)间Notch1表达强度差异无统计学意义(x2=0.631,P=0.427).Notch4、Jagged1以及Dll4的表达率在恶性黑素瘤与色素痣之间差异均有统计学意义(均P＜0.05),三者表达强度在原位与侵袭性黑素瘤之间差异亦均有统计学意义(P＜0.05).在黑素瘤组织中,Notch1与Jagged1的表达呈正相关(rs=0.350,P=0.027),与Dll4的表达亦呈正相关(rs=0.562,P=0.000),但是Jagged1与Dll4的表达呈负相关(rs=-0.734,P=0.000).结论 Notch信号通路异常可能是黑素瘤的发病机制之一,但具体作用机制有待进一步研究.     

恶性黑素瘤与磷脂酰肌醇3-激酶-Akt信号通路

磷脂酰肌醇3-激酶信号参与增殖、分化、凋亡和葡萄糖转运等多种细胞功能的调节.近年来发现,IA型磷脂酰肌醇3-激酶和其下游分子蛋白激酶B所组成的信号通路与人类恶性黑素瘤的发生发展密切相关.该通路调节黑素瘤细胞的增殖和存活,其活性异常不但能引起细胞恶性转化,而且与黑素瘤细胞的迁移、黏附、血管生成以及细胞外基质的降解等相关.目前以磷脂酰肌醇3-激酶-Akt信号通路关键分子为靶点的恶性黑素瘤治疗策略正在研究中.     

皮肤黑素瘤的分子检测和靶向治疗

黑素瘤的诊断和治疗是重要的临床课题。黑素瘤的组织学形态变异很多,与某些色素细胞痣之间存在中间型的组织形态学交叉,有时病理诊断相当困难。分子病理学的进展为皮肤黑素瘤的确诊提供了新的方法,同时也为黑素瘤的临床治疗提供了有价值的依据。BRAF,KIT,NRAS,HRAS等基因参与黑素瘤的发生和进展的MAPK通路和Akt/PI3K通路。DNA测序、外显子组测序、FISH技术、免疫组织化学技术可以检测黑素瘤相关的分子变化。针对黑素瘤发生分子通路的靶向治疗将使患者受益。     

黑素瘤相关DNA甲基化位点的初步筛查及异常甲基化谱的构建

【摘要】 目的 运用基因芯片技术筛查与黑素瘤相关的DNA异常甲基化位点，初步构建黑素瘤特异性甲基化谱。方法 采用Illumina Human Methylation 450K全基因组甲基化芯片对6例黑素瘤组织及其瘤旁组织标本进行全基因组DNA检测，得出差异DNA甲基化位点。采用Gene Ontology（GO）富集分析及KEGG_Pathway分析了解基因功能。结果 基因芯片检测结果显示，黑素瘤组织与瘤旁组织存在差异甲基化位点，共27 779个，其中16 673个为高甲基化位点，11 106个低甲基化位点。提高筛选条件为P < 0.01、︳Δβ︳ > 0.2，过滤掉所有单核苷酸多态性相关探针、位于XY染色体上的探针以及交叉反应的探针，共筛选得到4 883个差异甲基化位点，其中1 459（30%）个位于启动子区（包括TSS1500、TSS200、5′UTR、1st Exon）。GO富集分析显示，差异甲基化基因参与的生物学过程主要包括细胞生长、分化、黏附、运动迁移、信号转导及转录调控等。KEGG_Pathway分析显示，差异甲基化基因主要参与黏着斑、癌症通路、转化生长因子β信号通路、磷脂酰肌醇信号通路、黑素生成、趋化因子信号通路、黏合连接、钙信号通路、细胞黏附分子、MAPK信号通路、Wnt信号通路、JAK-STAT信号通路。基于“启动子区高甲基化位点对应的前16个基因、出现甲基化频率最高（CpG位点 ≥ 7）的基因、具有一定的功能或参与某条信号通路”条件，选出8个基因（KAAG1、DGKE、SOCS2、TFAP2A、GNMT、GALNT3、ANK2、HOXA9）作为黑素瘤候选生物标志物。结论 黑素瘤组织存在较多高甲基化基因，8个差异甲基化基因有可能作为黑素瘤的生物标志物。     

黑素瘤的分子发病机制和靶向治疗新进展

黑素瘤是一种皮肤恶性肿瘤,晚期患者预后不良,目前尚无有效的治疗方法。在黑素瘤中可以通过多种途径激活RAS/RAF/MEK/ERK(MAPK)和PI3K/AKT(AKT)两个信号传导通路。本文总结与黑素瘤发病机制最相关的信号传导通路及新的治疗策略。     

依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬

目的：研究依巴斯汀对人黑素瘤细胞自噬的影响及机制。方法：体外培养人黑素瘤细胞A375和M14,采用CCK-8增殖实验检测细胞活力,并计算IC50;利用mCherry-EGFP-LC3B双荧光指示系统检测自噬流;采用Western blot验证自噬相关蛋白LC3,Beclin1及信号通路蛋白的表达。结果：依巴斯汀可显著抑制人黑素瘤细胞的活力;依巴斯汀明显诱导人黑素瘤细胞中自噬小体和自噬溶酶体的产生;依巴斯汀显著上调人黑素瘤细胞中LC3-Ⅱ/Ⅰ的比值以及Beclin1的表达,同时抑制AKT/mTOR信号通路的活化,降低p-AKT/AKT和p-mTOR/mTOR。结论：依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬的发生。     

黑素瘤相关通路及治疗的新进展

黑素瘤的发病机制与遗传、环境因素如紫外线长期照射有关.研究表明,黑素瘤的发生发展主要与细胞外调节蛋白激酶通路和磷酸酰肌醇3激酶/蛋白激酶/哺乳动物雷帕霉素靶蛋白信号通路改变相关.相关的基因改变有:BRAF基因、NRAS基因、GNAQ基因和GNA 11基因、C-kit基因、苏氨酸蛋白激酶基因等.黑素瘤是皮肤侵袭性肿瘤,对于放疗和化疗均不敏感.针对黑素瘤发病机制的分子靶向治疗发挥了重要的作用,包括免疫治疗、基因治疗以及针对肿瘤血供的治疗.生物治疗比传统治疗有更好的靶向性和特异性.     

胶原三螺旋重复蛋白1、β联蛋白和Wnt5a在黑素瘤不同阶段的表达及相关性研究北大核心CSCD

目的探讨胶原三螺旋重复蛋白1（CTHRC1）、β联蛋白和Wnt5a在色素痣和黑素瘤不同发展阶段中的表达,探讨三者在黑素瘤发生、发展过程中的可能作用及相关性。方法免疫组化SP法检测23例色素痣、14例原位黑素瘤、21例侵袭性黑素瘤和18例转移性黑素瘤组织中CTHRC1、β联蛋白和Wnt5a蛋白的表达,结合临床病理特征进行分析。结果色素痣和黑素瘤组织中CTHRC1的阳性表达率分别为34．8％、62．3％,两者比较,差异有统计学意义（P〈0．05）;β联蛋白的阳性表达率分别为21．7％、62．3％,两者比较,差异有统计学意义（P〈0．05）;Wnt5a的阳性表达率分别为34．8％、69．8％,两者比较,差异有统计学意义（P〈0．05）。CTHRCl、13联蛋白的阳性表达与黑素瘤的浸润深度和淋巴结转移相关（P〈0．05）,wnt5a的阳性表达仅与淋巴结转移相关（P〈0．05）。黑素瘤中CTHRC1与β联蛋白之间以及CTHRC1与Wnt5a蛋白之间的阳性表达存在明显正相关（分别为r=0．798,P〈0．01;r=0．623,P〈0．01）。结论CTHRC1、β联蛋白和Wnt5a在黑素瘤的发生中起一定的作用,CTHRC1可能作为Wnt信号通路的辅助蛋白促进黑素瘤的发生和发展。     

表皮生长因子受体/丝氨酸/苏氨酸蛋白激酶通路对小鼠黑素瘤B16细胞迁移的影响

目的:研究表皮生长因子受体(EGFR)/丝氨酸/苏氨酸蛋白激酶(AKT)通路对小鼠黑素瘤B16细胞迁移的影响.方法:蛋白质免疫印迹方法分析各蛋白的表达;phagokinetic track motility法观察细胞的迁移.结果:表皮生长因子(EGF)能促进小鼠黑素瘤B16细胞迁移;AKT抑制剂(wortmannin)和水通道蛋白-3(aquaporins-3,AQP3)抑制剂(CuSO4)能抑制小鼠黑素瘤B16细胞迁移;EGF可诱导AKT磷酸化,EGF处理后5 min磷酸化AKT达到高峰.wortmannin和CuSO4可抑制细胞中AQP3的表达.结论:在小鼠黑素瘤B16细胞中,EGF通过磷酸化EGFR,激活AKT,进而使AQP3表达上调,促进B16细胞迁移.这一通路可被AKT和AQP3抑制剂阻断,这些涉及的信号通路可能成为潜在的治疗黑素瘤的靶点.     

ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达

目的检测ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达情况,初步探讨它们与黑素瘤发生、发展的关系。方法分别采用免疫组化和Western印迹法检测磷酸化的ERK1/2,JNK及P38MAPK在皮肤黑素瘤、皮内痣组织中的表达情况;用实时荧光定量聚合酶链反应(Real time-PCR)方法定量分析皮内痣和皮肤黑素瘤中ERK1/2,JNK和P38MAPK mRNA的表达情况。结果免疫组化和Western印迹法结果均显示p-ERK1/2,p-JNK及p-P38MAPK在皮肤黑素瘤中的表达高于皮内痣组(P0.05);Western印迹法显示p-ERK1/2,p-JNK及p-P38MAPK在皮内痣组中的表达高于正常对照组(P0.05)。ERK1/2,JNK及P38MAPK mRNA在皮肤黑素瘤和皮内痣中的表达均高于正常皮肤组织(P0.05);皮肤黑素瘤中ERK1/2,P38MAPKmRNA的表达均高于皮内痣(P0.05);而皮肤黑素瘤中JNK mRNA的表达与皮内痣组相比,差异无统计学意义(P0.05)。结论 ERK1/2,JNK和P38MAPK在皮肤黑素瘤表达增高,提示MAPK信号通路在皮肤黑素瘤发生、发展中起到重要作用,该信号通路有望成为预防和治疗皮肤黑素瘤的新靶点。     

黑素瘤相关微小RNA的研究进展

微小RNA参与细胞增殖、分化和凋亡是多种恶性肿瘤发生发展的重要调控因子.研究发现,多种微小RNA在黑素瘤中表达异常,其中Let-7家族、微小RNA 137、微小RNA 200c和微小RNA196a等发挥抑癌基因样作用,而微小RNA 221/222、微小RNA 30b/30d、微小RNA 146a、微小RNA 214和微小RNA 182发挥癌基因样作用.黑素瘤相关微小RNA的研究将丰富黑素瘤的分子生物学发病机制,为黑素瘤的早期诊断和治疗提供新的思路.     

Genetic 3′UTR variation is associated with human pigmentation characteristics and sensitivity to sunlight

Sunlight exposure induces signalling pathways leading to the activation of melanin synthesis and tanning response. MicroRNAs (miRNAs) can regulate the expression of genes involved in pigmentation pathways by binding to the complementary sequence in their 3′untranslated regions (3′UTRs). Therefore, 3′UTR SNPs are predicted to modify the ability of miRNAs to target genes, resulting in differential gene expression. In this study, we investigated the role in pigmentation and sun‐sensitivity traits, as well as in melanoma susceptibility, of 38 different 3′UTR SNPs from 38 pigmentation‐related genes. A total of 869 individuals of Spanish origin (526 melanoma cases and 343 controls) were analysed. The association of genotypic data with pigmentation traits was analysed via logistic regression. Web‐based tools for predicting the effect of genetic variants in microRNA‐binding sites in 3′UTR gene regions were also used. Seven 3′UTR SNPs showed a potential implication in melanoma risk phenotypes. This association is especially noticeable for two of them, rs2325813 in the MLPH gene and rs752107 in the WNT3A gene. These two SNPs were predicted to disrupt a miRNA‐binding site and to impact on miRNA‐mRNA interaction. To our knowledge, this is the first time that these two 3′UTR SNPs have been associated with sun‐sensitivity traits. We state the potential implication of these SNPs in human pigmentation and sensitivity to sunlight, possibly as a result of changes in the level of gene expression through the disruption of putative miRNA‐binding sites.     

miR‐137 inhibits proliferation of melanoma cells by targeting PAK2

MicroRNAs (miRNA) are key players in a variety of cancers including malignant melanoma. miR‐137 has been reported to be a tumor suppressor in melanoma and several targets have been identified for this miRNA. We previously developed a novel proteomics technology, ³⁵S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD). Because of its high sensitivity in analysing protein expression rates, SiLAD has the potential to unravel miRNA effects on mRNAs coding for proteins with long half‐lives or high abundance. Using SiLAD, we discovered that miR‐137 significantly downregulated the expression rate of p21‐activated kinase 2 (PAK2) in melanoma cells. Bioinformatics analysis predicted PAK2 as a direct target of miR‐137, which was confirmed by luciferase reporter assay and Western blot analysis. We found that overexpression of miR‐137 inhibited the proliferation of melanoma cells, which could be phenocopied by knockdown of PAK2 using siRNAs. Furthermore, overexpression of PAK2 restored miR‐137‐mediated suppression of cell proliferation. These findings indicate that miR‐137 could inhibit proliferation through targeting PAK2 in melanoma cells.     

Rising levels of serum S100 protein precede other evidence of disease progression in patients with malignant melanoma

BACKGROUND: Several serum markers may be useful in the detection of metastatic melanoma, but none is in routine clinical use. OBJECTIVE: To assess the validity of S100 protein as a serum marker of melanoma progression. METHODS: Serum S100 protein levels were measured in 496 serum samples from 214 melanoma patients, using the Sangtec luminescence immunoassay. There were 75 patients with stage 1 melanoma, 66 initially with stage 2 melanoma, 49 initially with stage 3 melanoma and 24 with stage 4 melanoma. RESULTS: Serum S100 protein levels were < 0.2 microg L-1 in 71 of 75 (95%) stage 1 patients. One patient who had a normal level developed local recurrence. Fifty-eight of 66 (88%) stage 2 patients also had normal serum S100 protein levels. One with elevated levels progressed to stage 3 melanoma and five with elevated levels progressed to stage 4 disease. The remaining two with elevated serum S100 protein remained well. Thirty-five of 49 (71%) stage 3 patients had normal levels and, of these, two have progressed to stage 4 disease. Three patients with stage 3 disease had an elevated serum S100 protein level on one occasion but remained well. Eleven of 13 patients who developed stage 4 melanoma during the study had rising levels of serum S100 protein > 0.2 microg L-1 5-23 weeks before detection of melanoma progression by conventional means. Twenty-two of 24 patients with stage 4 disease throughout the study had consistently elevated serum S100 protein levels, and the two patients with normal levels were clinically disease free after surgery and chemotherapy. None of 14 control subjects with atypical naevi had elevated S100 protein levels, and only one of 11 healthy normal controls had an elevated level. CONCLUSIONS: Thus, rising levels of serum S100 protein are a specific and sensitive clinically relevant marker of tumour progression in melanoma patients, which precedes other evidence of melanoma recurrence.     

胰岛素样生长因子ⅡmRNA结合蛋白3在恶性黑素瘤及良性痣中的表达

目的 探讨胰岛素样生长因子Ⅱ mRNA结合蛋白3(IMP3)在良性痣及黑素瘤组织中的表达,及其在恶性黑素瘤进展及诊断中的作用.方法 用IMP3抗体对28例恶性黑素瘤、8例Spitz痣、6例发育不良性痣和25例良性痣患者的标本组织进行免疫组化研究.结果 28例恶性黑素瘤组织标本中23例IMP3阳性,8例Spitz痣中4例阳性,6例发育不良性痣中2例阳性,25例良性痣均不表达.IMP3在黑素瘤中的表达明显高于Spitz痣及发育不良性痣(P<0.05),侵袭性黑素瘤表达明显高于原位黑素瘤(P<0.01).结论 IMP3可能是良性痣发展至恶性黑素瘤的一个生物学标志,在鉴别黑素瘤和良性痣之间存在一定的价值.     

Germline CDKN2A mutations among Greek patients with early-onset and multiple primary cutaneous melanoma

The genetic basis of melanoma susceptibility among Greek patients is uncharacterized. From 107 consecutive cutaneous melanoma patients, we analyzed the CDKN2A and CDK4 loci among 18 early-onset (< or =40 years) and two multiplex melanoma cases. Overall, we found three CDKN2A mutations (3/20; 15%), including one novel nonsense mutation (Trp110Stop) and two Arg24Pro missense alterations. There were no mutations in ARF or CDK4. CDKN2A mutations are not uncommon among Greek melanoma patients considering that none of the mutation carriers reported a family history of melanoma.     

Thermochemotherapy for malignant melanoma: combination therapy of ACNU and hyperthermia in mice

B16 melanoma (mouse melanoma) and C24 melanoma (human malignant melanoma) transplanted in mice were treated by a combined therapy of ACNU [1-(4-amino-2-methyl - 5 - pyrimidinyl) - methyl - 3 - (2 - chlorethyl) - 3 - nitrosourea hydrochloride] (10 mg/kg) and hyperthermia (43 degrees C, 30 min). In both types of melanoma, a marked synergistic effect of the combined therapy was noted. Particularly in C24 melanoma a reduction in the size of tumor was observed. Histopathologic findings revealed a strong degeneration such as destruction of the tumor structure and vacuolization of nuclei.     

Alteration of Human Melanoma Gangliosides by IFN-γ, IL-2, and IL-4

In lesions of malignant melanoma, melanoma cells are exposed to various cytokines produced by inflammatory reactions. As a result, transformation of melanoma cells is expected to occur. We studied alterations in human melanoma cell line ganglioside composition after exposing melanoma cell lines to interferon (IFN)-γ, interleukin (IL)-2, and IL-4 by biochemical methods. IFN-γ increases the ratio of a-series gangliosides and the ratio of GM3/GD3. This suggests an alteration of immunoreactivity, a decrease in ganglioside sialyltransferase II activity, and an decrease in the malignant character of these cells. The alteration of the ganglioside profile varied among cytokines and cell lines. The progression of malignant melanoma may be influenced by reciprocal interactions between the melanoma cells and the host immune system.     

TLR expression in human melanoma cells

There is increasing evidence that melanoma cells express TLR2, -3, and -4. However, the expression of other TLRs by these cells still remains unknown. We investigated the expression patterns of TLR2, -3, -4, -7, -8 and -9 both on melanoma-invaded lymph nodes and on melanoma cell lines. TLR2, -3, -4, -7 and -9 mRNA expression was determined by quantitative RT-PCR. The TLR protein expression level was measured ex vivo by immunohistochemistry and in vitro by flow cytometry. Results: At the mRNA level, melanoma cells in vitro, and possibly ex vivo, expressed TLR2, -3, -4, -7 and -9. TLR2 and -4 protein expressions ex vivo were over 50%, contrasting with an absence of these 2 TLRs in vitro. On the contrary, TLR-3 and -8 proteins had a low expression ex vivo with a high expression in vitro. TLR-7 and -9 proteins were expressed ex vivo and in vitro. Our study demonstrates for the first time that melanoma cells express TLR7 and -8.