Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs in patients with recurrent implantation failure

Abstract

N⁶-methyladenosine (m⁶A) is involved in most biological processes and actively participates in the regulation of reproduction. According to recent research, long non-coding RNAs (lncRNAs) and their m⁶A modifications are involved in reproductive diseases. In the present study, using m⁶A-modified RNA immunoprecipitation sequencing (m⁶A-seq), we established the m⁶A methylation transcription profiles in patients with recurrent implantation failure (RIF) for the first time. There were 1443 significantly upregulated m⁶A peaks and 425 significantly downregulated m⁶A peaks in RIF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that genes associated with differentially methylated lncRNAs are involved in the p53 signalling pathway and amino acid metabolism. The competing endogenous RNA network revealed a regulatory relationship between lncRNAs, microRNAs and messenger RNAs. We verified the m⁶A methylation abundances of lncRNAs by using m⁶A-RNA immunoprecipitation (MeRIP)-real-time polymerase chain reaction. This study lays a foundation for further exploration of the potential role of m⁶A modification in the pathogenesis of RIF.

Keywords: Long non-coding RNAs; M6A modified RNA immunoprecipitation sequencing; N6-methyladenosine; Recurrent implantation failure.

Fig. 1

The characteristics of m⁶A methylation in RIF. A A Venn diagram of m⁶A modification sites identified in lncRNAs from the RIF group (green) and the control group (blue). B A Venn diagram of m⁶A-modified lncRNAs from the RIF group (green) and the control group (blue). C The general number of differentially methylated peaks and associated lncRNAs; orange and blue represent hypermethylated and hypomethylated lncRNAs, respectively. D Cluster analysis of the m⁶A-modified lncRNA genes in the RIF and control groups. Red and green represent up- and downregulated peaks, respectively (fold-change ≥ 2.0, P < 0.00001). E The top five m⁶A motifs enriched in the RIF and control groups

Fig. 2

Distribution of differentially methylated m⁶A sites. A The volcano plot indicates the distribution of differential m⁶A peaks the in RIF. Red and blue represent up- and downregulated peaks, respectively (fold-change ≥ 2.0, P < 0.00001 with Fisher’ s exact test). B The positional relationship between differentially m⁶A methylated lncRNAs and mRNAs. Orange, DodgerBlue, olive, light grey, blue and green represent exon sense-overlap, intergenic, intronic antisense, natural antisense, intron sense-overlap and bidirectional, respectively. C The distribution of altered m⁶A peaks per lncRNA. Blue and orange represent down- and upregulated peaks, respectively. D The chromosomal distribution of all differentially methylated sites within lncRNAs. Blue and orange represent down- and upregulated peaks, respectively

Fig. 3

A and B The top 10 GO terms for genes associated with hyper- and hypomethylated lncRNAs, respectively. Orange indicates biological process, green indicates molecular function and blue indicates cellular component. C and D The top 5 KEGG pathways of genes associated with hyper- and hypomethylated lncRNAs, respectively

Fig. 4

Diagrams of the (A) phospholipase D and (B) p53 signalling pathways. These images have been reproduced from a previous publication [35] according to an open access licence

Fig. 5

The lncRNA–miRNA–mRNA regulatory network in RIF. The pink circular nodes represent upregulated lncRNAs, the purple circular nodes represent downregulated lncRNAs, the yellow triangular nodes represent miRNAs and the blue circular nodes represent mRNAs

Fig. 6

Validation of the m⁶A methylation abundances of lncRNAs. IGV plots of (A) ENST00000416361 and (B) ENST00000471664 m⁶A methylation and expression abundances in the RIF and control groups. Each plot shows the MeRIP-qPCR primer binding regions. At the bottom of the IGV plot, blue and red represent primer positions and the middle region is the pcr product region. IP refers to the methylation level, and input refers to the expression level. C The four-quadrant plot shows the inclusion of differentially methylated DEGs(Differentially Expressed Genes) for m⁶A peaks

Fig. 7

The m⁶A methylation abundances of (A) ENST00000416361 and (B) ENST00000471664 was validated in endometrial tissue with MeRIP-qPCR. The relative mRNA expression of (C) ENST00000416361 and (D) ENST00000471664 in endometrial tissue from the RIF group and Control group was determined with RT-qPCR. Statistical significance was determined using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001)