转录—代谢联合探讨色氨酸代谢通路在HER2阳性乳腺癌曲妥珠单抗耐药中的作用

Combined Transcription-Metabolism to Explore the Role of Tryptophan Metabolic Pathway in Trastuzumab Resistance in HER2-Positive Breast Cancer

关键词:  曲妥珠单抗耐药  色氨酸代谢  靶向代谢组学  实时荧光定量逆转录聚合酶链反应

Key words:  trastuzumab resistance  tryptophan metabolism  targeted metabolomics  real-time quantitative polymerase chain reaction(qRT-PCR)

摘要:

目的 探究人表皮生长因子受体2（human epidermal growth factor receptor 2, HER2）阳性乳腺癌曲妥珠单抗敏感细胞和耐药细胞在色氨酸通路中的代谢组学和转录组学差异。方法 采用基于实时荧光定量逆转录聚合酶链反应（real-time quantitative polymerase chain reaction, qRT-PCR）和超高效液相色谱-质谱联用（ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS）的靶向代谢组学方法检测色氨酸代谢中的代谢酶基因（AFMID、KYNU、AADAT、KYAT3、KYAT1、HAAO、KMO、CAT）和代谢物（喹啉酸、吡啶甲酸、朱砂酸、3-羟基犬尿氨酸、黄尿酸、犬尿酸、3-羟基邻氨基苯甲酸、烟酰胺、色氨酸、犬尿氨酸）在HER2阳性曲妥珠单抗敏感细胞和耐药细胞间的表达水平。采用正交偏最小二乘法判别分析（Orthogonal Partial Least-Squares Discrimination Analysis, OPLS-DA）法分别对代谢酶基因和代谢物的表达水平进行模式识别分析，以P < 0.05和差异倍数（Fold Change, FC）> 1.5或FC < 0.67为标准筛选差异代谢酶基因和差异代谢物，通过受试者工作特征曲线（Receiver Operating Characteristic curve, ROC）对差异代谢物和差异代谢酶基因的分类能力进行评估。结果 基于代谢酶基因和代谢物的表达水平建立的OPLS-DA模型能够区分敏感组细胞和耐药组细胞。相较于敏感组细胞，HAAO在耐药组中表达水平显著上调（P < 0.05），KMO和AFMID的表达水平在耐药组细胞中显著下调（P < 0.05），3-羟基邻氨基苯甲酸、犬尿氨酸、犬尿酸和朱砂酸的表达水平在耐药组细胞中显著上调（P < 0.05），烟酰胺的表达水平在耐药组细胞中显著下调（P < 0.05）。ROC分析发现犬尿氨酸、犬尿酸、朱砂酸、烟酰胺和KMO的曲线下面积（Area Under Curve, AUC）均大于0.8。结论 色氨酸代谢通路代谢酶基因KMO以及代谢物犬尿氨酸、犬尿酸、烟酰胺和朱砂酸在HER2阳性乳腺癌曲妥珠单抗耐药和敏感细胞中表达异常，有望为研究HER2阳性乳腺癌细胞曲妥珠单抗耐药的色氨酸代谢特征提供帮助。

Abstract:

OBJECTIVE To explore metabolomic and transcriptomic differences in the tryptophan pathway between trastuzumab-sensitive and drug-resistant cells in human epidermal growth factor receptor 2 (HER2) positive breast cancer. METHODS Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and targeted metabolomics basing on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) were used to detect levels of metabolic enzyme genes (AFMID, KYNU, AADAT, KYAT3, KYAT1, HAAO, KMO, CAT) and metabolites (quinolinic acid, picolinic acid, cinnabarinic acid, 3-hydroxykynurenine, xanthurenic acid, kynurenic acid, 3-hydroxyanthranilic acid, nicotinamide, tryptophan, kynurenine) in trastuzumab-sensitive cells and trastuzumab-resistant cells. Orthogonal Partial Least-Squares Discrimination Analysis (OPLS-DA) was used to analyze levels of metabolic enzyme genes and metabolites. The differential metabolic enzyme genes and metabolites were screened by the criteria of P < 0.05 and Fold Change (FC) > 1.5 or FC < 0.67. And the receiver operating characteristic curve (ROC) analysis was used to access the classification ability of differential metabolites and differential metabolic enzyme genes. RESULTS The OPLS-DA models based on the levels of metabolic enzyme genes and metabolites showed that trastuzumab-sensitive cells and trastuzumab-resistant cells have significant differences. Comparing to trastuzumab-sensitive cells, levels of HAAO were up-regulated in trastuzumab-resistant cells (P < 0.05) and levels of KMO and AFMID were down-regulated in trastuzumab-resistant cells (P < 0.05). Comparing to trastuzumab-sensitive cells, levels of 3-hydroxyanthranilic acid, kynurenine, kynurenic acid and cinnabarinic acid were up-regulated in trastuzumab-resistant cells (P < 0.05) and levels of nicotinamide were down-regulated in trastuzumab-resistant cells (P < 0.05). The ROC analysis result revealed that the Area Under Curve (AUC) of kynurenine, kynurenic acid, vincristine, nicotinamide and KMO were larger than 0.8. CONCLUSION Abnormal expression of the tryptophan metabolic pathway metabolizing enzyme gene KMO and metabolites kynurenine, kynurenic acid, nicotinamide, and cinnabarinic acid in trastuzumab-resistant and sensitive cells of HER2-positive breast cancers is expected to contribute to the study of tryptophan metabolic profiles of trastuzumab-resistant cells of HER2-positive breast cancers.