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.
Highlights
Post-transcriptional modifications of RNA has great research prospects and RNA epigenetics/epitranscriptomics has been proposed (He, 2010)
We revealed the presence of Gm and Um in human serum for the first time and quantified m6A, Gm, m1G, Cm, Um and m5U in serum from breast cancer patients and healthy controls
We demonstrated the differences of these modifications between breast cancer patients and healthy volunteers, and evaluate the potential of these monomethylated ribonucleosides as biomarkers for early detection of breast cancer
Summary
Post-transcriptional modifications of RNA has great research prospects and RNA epigenetics/epitranscriptomics has been proposed (He, 2010). Due to the functional identification of important regulatory proteins such as methyltransferase METTL3/ METTL14/WTAP complex (Liu et al, 2014) and demethylase FTO (Jia et al, 2011), RNA methylation modifications have attracted great attention and accumulating evidences have been obtained to confirm RNA methylation modifications as a novel layer of epigenetic alteration. Li et al reported that the level of Cm in serum is related to the reduced risk of developing esophageal squamous cell carcinoma (Li et al, 2021) These monomethylated ribonucleosides have significant potential to be used as indicators for early detection of cancers
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