MeCP2-induced alterations of transcriptome and m6A modification in retinal pigment epithelial cell

Authors: Zhang Yongya,  Li Xiaohua,  Zhao Xueru,  Li Xue
DOI: 10.3760/cma.j.cn115989-20230920-00105
Published 2024-05-10
Cite as Chin J Exp Ophthalmol, 2024, 42(5): 408-416.

Abstract                              [Download PDF] [Read Full Text]

Objective

To investigate mRNA and N6-methyladenosine (m6A) changes in retinal pigment epithelium (RPE) cells treated with recombinant human methyl-CpG binding protein 2 (MeCP2) and the mechanisms.

Methods

The passaged ARPE-19 cells were divided into normal control and MeCP2 groups after adhesion culture.Cells in the normal control group were continuously cultured in normal culture medium, and the cells in the MeCP2 group were cultured in culture medium containing a final concentration of 20 ng/ml of recombinant human MeCP2 protein for 72 hours.Transcriptomic sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were used to extract and analyze total RNA.Differentially methylated genes (DMGs) and differentially expressed genes (DEGs) were screened using the edgeR software package based on P<0.05.The biological function of differential genes was determined by gene ontology (GO) enrichment analysis, and the pathway enrichment analysis was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG).Intersection of genes between DEGs and DMGs were screened, and real-time fluorescence quantitative PCR was used to determine the mRNA expression levels of differential genes.

Results

A total of 100 DEGs and 7 441 DMGs genes were screened.According to enrichment analysis, the DEGs were enriched to extracellular matrix (ECM)-receptor interaction, cell division, phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway and so on.The DMGs were associated with microtubule cytoskeleton, angiogenesis, epidermal growth factor receptor (ErbB) signaling pathway, advanced glycation end-products (AGEs) -glycation end-products receptor (RAGE) signaling pathway, mammalian target of rapamycin (mTOR) signaling pathway, Notch signaling pathway and transforming growth factors-β (TGF-β) signaling pathway and so on.There were 24 up-regulated and 76 down-regulated DEGs.Five DMGs had hypermethylation peaks, and 7 439 DMGs had hypomethylation peaks.After annotation of peaks, 7 626 genes in the normal control group and 8 006 genes in the MeCP2 group had m6A methylation, with 7 360 intersecting genes between the two groups.The m6A methylation in the normal control group and MeCP2 group was concentrated in the CDS, intron and 3′-untranslated region (3’UTR) regions of the transcript, with the methylation ratio of 23.62%/22.27%, 48.53%/48.35% and 23.66%/25.28%, respectively.Joint analysis showed that CSPG5 and RBP1 genes related to the epithelial-mesenchymal transition (EMT) had lower amount of mRNA and m6A.Fluorescence quantitative PCR results showed that the relative mRNA expression levels of GSPG5RBP1 and ZNF484 in MeCP2 group were significantly lower than those in normal control group (t=7.885, 7.613, 7.345; all at P<0.01).

Conclusions

The regulatory mechanism of MeCP2 on EMT in RPE cells is related to m6A methylation modification.CSPG5 and RBP1 genes may be the target genes of m6A methylation and participate in the EMT regulated by MeCP2.

Key words:

Proliferative vitreoretinopathy; Retinal pigment epithelial cells; MeCP2; m6A; MeRIP-seq; Epithelial-mesenchymal transition

Contributor Information

Zhang Yongya

Department of Ophthalmology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Key Laboratory of Ophthalmology and Visual Science, Henan Academy of Innovations in Medical Science, Zhengzhou 450003, China

Li Xiaohua

Department of Ophthalmology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Key Laboratory of Ophthalmology and Visual Science, Henan Academy of Innovations in Medical Science, Zhengzhou 450003, China

Zhao Xueru

Department of Ophthalmology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Key Laboratory of Ophthalmology and Visual Science, Henan Academy of Innovations in Medical Science, Zhengzhou 450003, China

Li Xue

Department of Ophthalmology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Key Laboratory of Ophthalmology and Visual Science, Henan Academy of Innovations in Medical Science, Zhengzhou 450003, China

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