Comprehensive Analysis of 5-Methylcytosine Profiles of Messenger RNA in Human High-Grade Serous Ovarian Cancer by MeRIP Sequencing

Abstract

Background Accumulating evidence has indicated that methylation status is closely related to tumourigenesis and a few aggressive features of diverse cancers. However, as an important methylation regulation modification, the distribution of 5-methylcytosine (m5C) in high-grade serous ovarian cancer (HGSOC) remains unclear. Materials and Methods We collected three pairs of human HGSOC tissues and adjacent non-tumour tissues to analyse the transcriptome-wide m5C methylation of messenger RNAs (mRNAs) by methylated RNA immunoprecipitation sequencing. Gene ontology (GO) enrichment analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis were performed to eExplore the potential biological functions of these genes and important cancer pathways. We used immunohistochemistry to analyse the expression of the m5C modification regulatory gene MAP2K3 in 80 HGSOC tissue samples, and their associations with clinical parameters were analyzed using the Spearman-rho test. Univariate and multivariate Cox regression analyses were performed to identify potential prognostic factors. Kaplan–Meier analysis was performed to analyze overall survival. Results We identified 2050 dysregulated m5C peaks, 1767 of which were significantly upregulated, while 283 were significantly downregulated. GO enrichment analysis showed that genes altered by the m5C peak played a key role in system development, transporter complex, and transporter activity. KEGG pathway analysis revealed that these genes were enriched in some important pathways in cancer regulation, such as inflammatory mediator regulation of the TRP channels pathway, Wnt signalling pathway, and focal adhesion pathways. In addition, through joint analysis of MeRIP-seq and RNA-seq data, we identified 125 differentially methylated m5C peaks and synchronous differentially expressed genes. These genes play key roles in cell growth, maintenance, plasma membranes, and cell adhesion molecule activity. Immunohistochemical staining results showed that high expression of MAP2K3 was significantly correlated with CA125 level (p < 0.001), tumour size (p = 0.001), lymph node metastasis (p = 0.008), depth of myometrial invasion (p < 0.001), and FIGO stage (p < 0.001), indicating a poor prognosis. Conclusion Our results reveal the different distribution patterns of m5C in HGSOC and adjacent tissues and the possible involvement of m5C in HGSOC cell functions. Our study provides new insights into the epi-transcriptomic dysregulation of m5C in the tumourigenesis of HGSOC.

Overview of distinct 5-methylcytosine profiles of messenger RNA in normal and knock-down NSUN2 colorectal cancer cells

“…The number of m5C modification peaks detected in this study is also approximately within the above range. The number of m5C modification peaks detected in the other two studies ( Zhang et al, 2020 ; Meng et al, 2021 ) is also approximately consistent with this study. The research team at Sun Yat-sen University conducted transcriptome sequencing analysis of 7 human tissues and 10 mouse tissues, identifying a total of 3,212 and 2,498 high confidence sites ( Huang et al, 2019 ).…”

Biological roles of RNA m5C modification and its implications in Cancer immunotherapy

“…Naïve B cells, CD8 + T cells, Treg cells, and resting NK cells had a higher expression level in the low-risk group while the M0 and M2 phenotype macrophages had a higher expression level in the highrisk group, suggesting that m 5 C-related lncRNAs may regulate pancreatic cancer progression via promoting M2 phenotype macrophage polarization or infiltration in PDAC [116]. Related expression also exists in the research of Head and Neck Squamous Cell Carcinoma [117], hepatocellular carcinoma [118], breast cancer [119], human high-grade serous ovarian cancer [120], cutaneous melanoma [121] and colon cancer [122].…”

Comprehensive analysis of transcriptomic profiling of 5‐methylcytosin modification in placentas from preeclampsia and normotensive pregnancies

“…39 Whether in High-grade serous ovarian cancer (HGSOC) tissues or paired normal tissues, the m5C was both mainly enriched in the coding sequence, and a distinct peak was observed in the initiation region of CDS and an obvious drop in the vicinity of the stop codon of CDS. 40 Similarly, the m5C methylation in the placental tissue whether from normal pregnancy or preeclampsia were found to be mainly enriched in coding regions, especially with a remarkable peak after the start codon of 5′UTR, but significantly drop before the stop code of 3′UTR. However, compared with normal controls, the peaks of methylated cytosines in the placentas of PE patients were remarkably increased before the stop code of CDS and even higher than the peak value after start code in CDS.…”

“…Specifically, m5C is mostly distributed in the vicinity of stop codon and least distributed at 5′UTR in both groups 39 . Whether in High‐grade serous ovarian cancer (HGSOC) tissues or paired normal tissues, the m5C was both mainly enriched in the coding sequence, and a distinct peak was observed in the initiation region of CDS and an obvious drop in the vicinity of the stop codon of CDS 40 . Similarly, the m5C methylation in the placental tissue whether from normal pregnancy or preeclampsia were found to be mainly enriched in coding regions, especially with a remarkable peak after the start codon of 5′UTR, but significantly drop before the stop code of 3′UTR.…”