Comprehensive Analysis of the Transcriptome-Wide m6A Methylome in Endometrioid Ovarian Cancer

Abstract

Emerging studies have revealed that N6-methyladenosine modification is involved in the development of various cancers. However, the m6A modification pattern of endometrioid ovarian cancer (EOC) has not been demonstrated. In the present study, high-throughput sequencing combined with methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing were used to obtain the transcriptome-wide m6A modifications of endometrioid ovarian cancer for the first time. The roles of methyltransferase-like 3 (METTL3) in EOC cell line COV362 were explored. In total, 39,237 m6A-modified peaks related to 17,082 genes were identified in the EOC group, and 52,848 m6A peaks representing 19,349 genes were detected in endometriosis group. Functional enrichment analysis revealed that m6A enriched genes were associated with tight junctions, cell adhesion molecules, platinum drug resistance, adherens junction, and more. METTL3 knockdown in the COV362 cells significantly decreased cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G0/G1 phase. Our study presented the transcriptome-wide m6A modifications of endometrioid ovarian cancer for the first time and revealed various differentially expressed genes with methylated m6A modifications. This study may provide new directions for in-depth research of the underlying molecular mechanisms and signaling pathways of EOC development and progression.

Keywords: METTL3; MeRIP-seq; N6-methyladenosine; endometrioid ovarian cancer; modification patterns.

Figure 1

Characteristics of m6A distributions in EOC and OE. (A) Venn diagrams of overlaps of m6A peaks and (B) genes in EOC and OE tissues. (C) The distribution of differentially modified m6A peaks with significance per gene. (D) Number of peaks per gene in EOC and OE tissues. (E) Volcano plots showing the significant differential m6A peaks in EOC samples compared with OE samples. (F) The distribution and difference in the density of m6A peaks along all the transcripts in EOC and OE. (G) Pie charts showing the distributions of m6A peaks in EOC and OE tissues. (H) Representative m6A motifs enriched from the altered m6A peaks in EOC and OE groups, with P-values of 1*e^-166 and 1*e^-247, respectively.

Figure 2

GO and KEGG pathway enrichment of hyper-methylated m6A genes. (A) Major enrichment and meaningful GO terms of hyper-methylated m6A genes in EOC. (B) The top 20 significant GO enrichment terms with m6A hypermethylation. (C) The top 20 significant KEGG pathways with m6A hypermethylation.

Figure 3

Conjoint analysis of MeRIP-seq and RNA sequencing data. (A) Scatter plot presenting the differentially expressed genes in EOC. (B) Four-quadrant plots presenting the distribution of genes with significant alterations in both the m6A modification and mRNA levels. (C) Heatmap plots exhibiting the differentially expressed genes of EOC and OE groups.

Figure 4

GO terms and KEGG pathways of genes with significant alterations in both the m6A modification and mRNA levels. (A) Major enrichment and meaningful GO terms of genes with significant alterations in both the m6A modification and mRNA levels. (B) The top 20 GO terms of genes with significant alterations in both the m6A modification and mRNA levels. (C) The top 10 KEGG pathways of genes with significant alterations in both the m6A modification and mRNA levels.

Figure 5

(A) The relative mRNA expression level of METTL3 in EOC and OE groups. (B) The relative mRNA expression level of KRT8 in EOC and OE groups. (C) Real-time PCR analysis and western blot analysis for METTL3 expression in COV362 untreated cells (Control), cells transfected with empty vector (Vector), and cells transfected with METTL3-siRNA (METTL3-siRNA). (D) MTT assay showing cell viability of Control, Vector and METTL3-siRNA cells. (E) The TUNEL assay showing the apoptosis rates of Control, Vector and METTL3-siRNA cells. (F) Cell cycle analysis of Control, Vector and METTL3-siRNA cells. (G) Western blot showing the expression levels of KRT8 and FAS in Control, Vector and METTL3-siRNA cells. * P < 0.05, ** Compared with the other groups, P < 0.01.