Mass Spectrometry-Based Targeted Serum Monomethylated Ribonucleosides Profiling for Early Detection of Breast Cancer

Zhihao Fang; Yiqiu Hu; Jiani Chen; Kailun Xu; Kailai Wang; Shu Zheng; Cheng Guo

doi:10.3389/fmolb.2021.741603

Mass Spectrometry-Based Targeted Serum Monomethylated Ribonucleosides Profiling for Early Detection of Breast Cancer

Front Mol Biosci. 2021 Aug 26:8:741603. doi: 10.3389/fmolb.2021.741603. eCollection 2021.

Authors

Zhihao Fang¹, Yiqiu Hu¹, Jiani Chen¹, Kailun Xu¹, Kailai Wang¹, Shu Zheng^{1

2}, Cheng Guo^{1

2}

Affiliations

¹ Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
² Cancer Center, Zhejiang University, Hangzhou, China.

Abstract

RNA methylation plays a significant regulatory role in various of physiological activities and it has gradually become a hotspot of epigenetics in the past decade. 2'-O-methyladenosine (Am), 2'-O-methylguanosine (Gm), 2'-O-methylcytidine (Cm), 2'-O-methyluridine (Um), N ⁶-methyladenosine (m⁶A), N ¹-methylguanosine (m¹G), 5-methylcytidine (m⁵C), and 5-methyluridine (m⁵U) are representative 2'-O-methylation and base-methylation modified epigenetic marks of RNA. Abnormal levels of these ribonucleosides were found to be related to various diseases including cancer. Serum is an important source of biofluid for the discovery of biomarkers, and novel tumor biomarkers can be explored by measuring these ribonucleoside modifications in human serum. Herein, we developed and applied a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method to determine the content of monomethylated ribonucleosides in human serum. The developed method enabled sensitive and accurate determination of these monomethylated ribonucleosides. By applying this robust method, we demonstrated the presence of Gm and Um in human serum for the first time, and we successfully quantified m⁶A, Gm, m¹G, Cm, Um and m⁵U in serum samples collected from 61 patients with breast cancer and 69 healthy controls. We discovered that the levels of Gm, m¹G, Cm, Um and m⁵U in serum were all significantly decreased in breast cancer patients whereas m⁶A was increased. We performed receiver operating characteristic (ROC) curve analysis, and obtained highest area under curve (AUC) value when combining these six monomethylated ribonucleosides together. These results suggest that m⁶A, Gm, m¹G, Cm, Um and m⁵U might have great potential to be novel biomarkers for detection of breast cancer in the early stage. In addition, this study may stimulate future investigations about the regulatory roles of monomethylated ribonucleosides on the initiation and development of breast cancer.

Keywords: HILIC-MS/MS; RNA modification; breast cancer; monomethylated ribonucleosides; serum.