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. 2020 Sep 15:8:e9880.
doi: 10.7717/peerj.9880. eCollection 2020.

Integrated analysis of the transcriptome-wide m6A methylome in preeclampsia and healthy control placentas

Jin Wang  1 , Fengchun Gao  2 , Xiaohan Zhao  1 , Yan Cai  1 , Hua Jin  1
Affiliations

Integrated analysis of the transcriptome-wide m6A methylome in preeclampsia and healthy control placentas

Jin Wang et al. PeerJ. .

Abstract

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic mRNA and potential regulatory functions of m6A have been shown by mapping the RNA m6A modification landscape. m6A modification in active gene regulation manifests itself as altered methylation profiles. The number of reports regarding to the profiling of m6A modification and its potential role in the placenta of preeclampsia (PE) is small. In this work, placental samples were collected from PE and control patients. Expression of m6A-related genes was investigated using quantitative real-time PCR. MeRIP-seq and RNA-seq were performed to detect m6A methylation and mRNA expression profiles. Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were also conducted to explore the modified genes and their clinical significance. Our findings show that METTL3 and METTL14 were up-regulated in PE. In total, 685 m6A peaks were differentially expressed as determined by MeRIP-seq. Altered peaks of m6A-modified transcripts were primarily associated with nitrogen compound metabolic process, positive regulation of vascular-associated smooth muscle cell migration, and endoplasmic reticulum organisation. The m6A hyper-methylated genes of Wnt/β-catenin signalling pathway, mTOR signalling pathway, and several cancer-related pathways may contribute to PE. We also verified that the significant increase of HSPA1A mRNA and protein expression was regulated by m6A modification, suggesting m6A plays a key role in the regulation of gene expression. Our data provide novel information regarding m6A modification alterations in PE and help our understanding of the pathogenesis of PE.

Keywords: m6A; Preeclampsia; RNA methylation.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. METTL3 and METTL14 were up-regulated in PE.
Quantitative real-time PCR was used to analysis the mRNA levels of METTL3, METTL14, FTO, WTAP, and ALKBH5 in the PE samples and samples from normal pregnant women. All p-values were calculated using Student’s t-test. ∗p < 0.05 versus control group (n = 4 each). PE, preeclampsia.
Figure 2
Figure 2. m6A levels of total RNA in PE and control.
m6A levels of total RNA were determined by antibody based colorimetric method. Data are presented as mean ± SD. All p-values were calculated using Student’s t-test. ∗∗p < 0.01 versus control group (n = 4 each). PE, preeclampsia.
Figure 3
Figure 3. Overview of the m6A methylation landscape in the preeclampsia and control samples.
(A) Volcano plots displaying the distinct m6A peaks and their statistical significance (fold changes ≥1.5 and p < 0.05). (B) Metagene plots showing the region of average m6A peaks throughout the transcripts in the preeclampsia and control samples. (C) Pie charts displaying the distribution of m6A peaks in the preeclampsia. (D) Pie charts displaying the distribution of m6A peaks in the control group. (E) Sequence motifs of the m6A-containing peak regions. (F) Data visualisation analysis of HSPA1A mRNA m6A modifications in the preeclampsia group. (G) Data visualisation analysis of HSPA1A mRNA m6A modifications in the control group.
Figure 4
Figure 4. Gene ontology and KEGG pathway analyses of the altered m6A transcripts.
(A) Major enriched and significant GO-assessed up-regulated m6A peak transcripts. (B) Major enriched and significant GO-assessed down-regulated m6A peaks transcripts. (C) The top ten significantly enriched pathways for the up-regulated m6A peaks transcripts. (D) The top ten significantly enriched pathways for the down-regulated m6A peaks transcripts. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cellular component; MF, molecular function.
Figure 5
Figure 5. Conjoint analysis of m6A-RIP-seq and RNA-sequencing data for preeclampsia and control samples.
(A) Volcano plots displaying the mRNAs that were differentially expressed between the preeclampsia and control groups and their statistical significance (fold changes ≥1.5 and p < 0.05). (B) Hierarchical clustering analysis of the differentially expressed mRNAs. (C) Four quadrant graph showing the distribution of transcripts with a significant change in both m6A level and expression in preeclampsia.
Figure 6
Figure 6. The expression of HSPA1A is regulated by m6A modification.
(A) Schematic presentation of the location of verified m6A site in HSPA1A (B) The m6A levels of HSPA1A at the particular site in the CDS region were significantly upregulated in PE. (C) The mRNA levels of HSPA1A were significantly increased in PE. (D) HSP70 protein expression was assessed by Western blot. All p-values were calculated using Student’s t-test. ∗p < 0.05 versus control group (n = 4 each). PE, preeclampsia.
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Grants and funding

This research was funded by the Natural Science Foundation of Shandong Province (No. ZR2014HP047), the Science and Technology Development Project of Jinan (No. 201907013), and the Science and Technology Project of Jinan Health Committee (No. 2019-1-33). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.