Arginine decarboxylase （ADC） plays an important role in polyamine biosynthesis in plants. In order to study the structure and expression ofADC gene in mango, an arginine decarboxylase gene ADC, named as MiADC, was c...Arginine decarboxylase （ADC） plays an important role in polyamine biosynthesis in plants. In order to study the structure and expression ofADC gene in mango, an arginine decarboxylase gene ADC, named as MiADC, was cloned and isolated from the tender leaves of mango using RACE technology, and the expression of MiADC was analyzed by qRT-PCR based on the cDNA sequence of MiADC gene in this study. The results showed that the full length of MiADC was 3 089 bp containing a 575 bp 5＇-UTR, a 2 178 bp ORF with file initial codon and the terminal codon, a 311 bp 3＇-UTR and a 25 bp poly （A）; and file MiADC had no intron. The homologous analysis in the NCBI demonstrated that the sequence of MiADC had the highest similarity at 78% to Poncirus trifoliata arginine decarboxylase gene （HQ008237.1）, and the prediction protein also had the highest homology at 78% level with Poncirus trifoliata arginine decarboxylase （AEE99192.1） . Phylogenetic tree analysis revealed that the MiADC in mango was clustered in the same clade as the arginine decarboxylase from Poncirus trifoliate. Expression analysis showed that the MiADC gene was expressed in roots, stems, leaves, flowers, fruitlet, ripe fruit, and the transcriptional level of the cloned gene was affected obviously by low temperature, which proved that the MiADC gene may be a low temperature stress responding gene.展开更多
目的:通过芯片分析联合网络药理学及其实验验证,阐明淫羊藿干预乳腺癌干细胞(BCSCs)的潜在分子标志物和药物-化合物-靶点机制。方法:在中药系统药理学数据库与分析平台(TCMSP)检索相关药物信息,获得淫羊藿的活性成分和潜在靶点。在基因...目的:通过芯片分析联合网络药理学及其实验验证,阐明淫羊藿干预乳腺癌干细胞(BCSCs)的潜在分子标志物和药物-化合物-靶点机制。方法:在中药系统药理学数据库与分析平台(TCMSP)检索相关药物信息,获得淫羊藿的活性成分和潜在靶点。在基因表达综合(GEO)数据库检索"Breast Cancer Stem Cells",通过分析与筛选,获取GSE98239芯片数据,使用GEO2R在线分析工具获取其差异基因,绘制差异基因热图和火山图。通过Cytoscape 3.8.0构建淫羊藿干预乳腺癌干细胞差异基因网络图,并进行药物与疾病基因的基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析。将人乳腺癌MDAMB-231细胞分为20%,40%,60%淫羊藿含药血清组和对照组,通过细胞增殖与活性检测(CCK-8),蛋白免疫印迹法(Western blot)检测淫羊藿含药血清干预MDA-MB-231后,对其活性和乳腺癌细胞中靶蛋白表达量的影响。结果:获得淫羊藿含有的黄酮、甾醇、生物碱及倍半萜类活性成分23个,发现其与乳腺癌干细胞中的B细胞淋巴瘤-2样蛋白1(BCL2L1),基质金属蛋白酶2(MMP2),人前列腺素内过氧化物合酶2(PTGS2),血管内皮生长因子(VEGF)A,转化生长因子β受体Ⅰ(TGFBR1)等枢纽基因相互作用,参与诱导新的血管生成、细胞迁移,使BCSCs持续自我更新而凋亡减少并发生细胞迁移,从而促进乳腺癌的复发和转移。KEGG结果显示,淫羊藿干预pathway in cancer信号通路中转化生长因子β(TGF-β),VEGF,胞内磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt),丝裂原活化蛋白激酶(MAPK),哺乳动物雷帕霉素靶蛋白(mTOR)亚通路的多个差异表达基因(DEGs)来发挥其干预乳腺癌干细胞的功效。实验表明,淫羊藿含药血清干预后乳腺癌细胞存活率显著降低,乳腺癌细胞中TGFBR1和Smad2表达量显著降低(P<0.01)。结论:淫羊藿不同浓度含药血清中的多个组分,能够协同作用于乳腺癌干细胞的目标差异表达基因,并通过下调TGF-β通路中关键分子TGFBR1及下游信号Smad2蛋白的表达水平,从而发挥抑制乳腺癌细胞增殖的作用。展开更多
文摘Arginine decarboxylase （ADC） plays an important role in polyamine biosynthesis in plants. In order to study the structure and expression ofADC gene in mango, an arginine decarboxylase gene ADC, named as MiADC, was cloned and isolated from the tender leaves of mango using RACE technology, and the expression of MiADC was analyzed by qRT-PCR based on the cDNA sequence of MiADC gene in this study. The results showed that the full length of MiADC was 3 089 bp containing a 575 bp 5＇-UTR, a 2 178 bp ORF with file initial codon and the terminal codon, a 311 bp 3＇-UTR and a 25 bp poly （A）; and file MiADC had no intron. The homologous analysis in the NCBI demonstrated that the sequence of MiADC had the highest similarity at 78% to Poncirus trifoliata arginine decarboxylase gene （HQ008237.1）, and the prediction protein also had the highest homology at 78% level with Poncirus trifoliata arginine decarboxylase （AEE99192.1） . Phylogenetic tree analysis revealed that the MiADC in mango was clustered in the same clade as the arginine decarboxylase from Poncirus trifoliate. Expression analysis showed that the MiADC gene was expressed in roots, stems, leaves, flowers, fruitlet, ripe fruit, and the transcriptional level of the cloned gene was affected obviously by low temperature, which proved that the MiADC gene may be a low temperature stress responding gene.
文摘目的:通过芯片分析联合网络药理学及其实验验证,阐明淫羊藿干预乳腺癌干细胞(BCSCs)的潜在分子标志物和药物-化合物-靶点机制。方法:在中药系统药理学数据库与分析平台(TCMSP)检索相关药物信息,获得淫羊藿的活性成分和潜在靶点。在基因表达综合(GEO)数据库检索"Breast Cancer Stem Cells",通过分析与筛选,获取GSE98239芯片数据,使用GEO2R在线分析工具获取其差异基因,绘制差异基因热图和火山图。通过Cytoscape 3.8.0构建淫羊藿干预乳腺癌干细胞差异基因网络图,并进行药物与疾病基因的基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析。将人乳腺癌MDAMB-231细胞分为20%,40%,60%淫羊藿含药血清组和对照组,通过细胞增殖与活性检测(CCK-8),蛋白免疫印迹法(Western blot)检测淫羊藿含药血清干预MDA-MB-231后,对其活性和乳腺癌细胞中靶蛋白表达量的影响。结果:获得淫羊藿含有的黄酮、甾醇、生物碱及倍半萜类活性成分23个,发现其与乳腺癌干细胞中的B细胞淋巴瘤-2样蛋白1(BCL2L1),基质金属蛋白酶2(MMP2),人前列腺素内过氧化物合酶2(PTGS2),血管内皮生长因子(VEGF)A,转化生长因子β受体Ⅰ(TGFBR1)等枢纽基因相互作用,参与诱导新的血管生成、细胞迁移,使BCSCs持续自我更新而凋亡减少并发生细胞迁移,从而促进乳腺癌的复发和转移。KEGG结果显示,淫羊藿干预pathway in cancer信号通路中转化生长因子β(TGF-β),VEGF,胞内磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt),丝裂原活化蛋白激酶(MAPK),哺乳动物雷帕霉素靶蛋白(mTOR)亚通路的多个差异表达基因(DEGs)来发挥其干预乳腺癌干细胞的功效。实验表明,淫羊藿含药血清干预后乳腺癌细胞存活率显著降低,乳腺癌细胞中TGFBR1和Smad2表达量显著降低(P<0.01)。结论:淫羊藿不同浓度含药血清中的多个组分,能够协同作用于乳腺癌干细胞的目标差异表达基因,并通过下调TGF-β通路中关键分子TGFBR1及下游信号Smad2蛋白的表达水平,从而发挥抑制乳腺癌细胞增殖的作用。
