背景:我们研究了黑色素瘤细胞中黑色素发生和抗凋亡相关的黑色素瘤因子(TXM1,TXM18,A375P,和A375SM)。
目的:寻找黑素瘤相关中枢因子,研究了与生物信息学集成的基于高通量筛选的技术。
方法:用商业系统进行阵列CGH分析。用Cy3-dCTP和Cy5-dCTP适当地标记从具有对照DNA的每个细胞系单独制备的总基因组DNA并杂交,随后通过log2绿色(G;测试)与红色(R;参考)荧光比(G/R)进行数据处理。通过具有log2转化比率的斑点来判断拷贝数的增益或损失。使用STRING数据库中的人相互作用组进行基于阵列CGH结果的检测到的候选基因的PPI定位分析。使用CHARMM中的隐式溶剂化模型进行能量最小化和短分子动力学(MD)模拟,以分析YWHAZ和YWHAB之间的相互作用残基。
结果:三个基因(BMP-4,BFGF,LEF-1)已知与黑素生成有关的染色体拷贝数丢失,Chr.6q23.3在所有测试的细胞系中丢失。十个hub基因(CTNNB1、PEX13、PEX14、PEX5、IFNG、EXOSC3,EXOSC1,EXOSC8,UBC,和PEX10)被预测为6q23.3基因座网络中的功能相互作用因子。细胞凋亡相关基因E2F1、p50、BCL2L1和BIRC7,和FGF2在测试的黑色素瘤细胞系中丢失了染色体拷贝数。YWHAB,获得了染色体拷贝数,被预测为黑色素瘤细胞中最重要的hub蛋白。进行了结合YWHAB和YWHAZ的分子动力学模拟,预测该复合物通过其3个氢键模式在能量和结构上是稳定的。相互作用残基的数目是27。
结论:我们的研究比较了全基因组筛查对黑素瘤因素的预测,并为了解黑素瘤黑色素发生和抗凋亡相关机制提供了新的信息。尤其是,YWHAB在黑色素瘤细胞中被新检测为核心因子。
BACKGROUND: We investigated melanogenesis- and anti-apoptosis-related melanoma factors in melanoma cells (TXM1, TXM18, A375P, and A375SM).
OBJECTIVE: To find melanoma associated hub factor, high-throughput screening-based techniques integrating with bioinformatics were investigated.
METHODS: Array CGH analysis was conducted with a commercial system. Total genomic DNAs prepared individually from each cell line with control DNA were properly labeled with Cy3-dCTP and Cy5-dCTP and hybridizations and subsequently performed data treatment by the log2 green (G; test) to red (R; reference) fluorescence ratios (G/R). Gain or loss of copy number was judged by spots with log2-transformed ratios. PPI mapping analysis of detected candidate genes based on the array CGH results was conducted using the human interactome in the STRING database. Energy minimization and a short molecular dynamics (MD) simulation using the implicit solvation model in CHARMM were performed to analyze the interacting residues between YWHAZ and
YWHAB.
RESULTS: Three genes (BMP-4, BFGF, LEF-1) known to be involved in melanogenesis were found to lose chromosomal copy numbers, and Chr. 6q23.3 was lost in all tested cell lines. Ten hub genes (CTNNB1, PEX13, PEX14, PEX5, IFNG, EXOSC3, EXOSC1, EXOSC8, UBC, and PEX10) were predicted to be functional interaction factors in the network of the 6q23.3 locus. The apoptosis-associated genes E2F1, p50, BCL2L1, and BIRC7 gained, and FGF2 lost chromosomal copy numbers in the tested melanoma cell lines.
YWHAB, which gained chromosomal copy numbers, was predicted to be the most important hub protein in melanoma cells. Molecular dynamics simulations for binding
YWHAB and YWHAZ were conducted, and the complex was predicted to be energetically and structurally stable through its 3 hydrogen-bond patterns. The number of interacting residues is 27.
CONCLUSIONS: Our study compares genome-wide screening interactomics predictions for melanoma factors and offers new information for understanding melanogenesis- and anti-apoptosis-associated mechanisms in melanoma. Especially,
YWHAB was newly detected as a core factor in melanoma cells.