Abstract Body: The lymphatic endothelial cell transcriptomic response to stimuli is dynamic and requires precise coordination. Post-transcriptional Adenosine methylation (m6A) of mRNA can greatly impact mRNA stability and subsequent protein abundance but are understudied in regulation of lymphatic physiological processes. In this work we test the hypothesis that the canonical lymphangiogenic cytokine VEGFC drives m6A mRNA methylation in human dermal LEC (hdLEC).
VEGFC stimulation of cultured primary hdLEC induced expression of several m6A reader, writer, and eraser proteins. Nanopore RNA sequencing of mRNA isolated from VEGFC-stimulated hdLEC indicated that VEGFC-stimulation modulated m6A marking of mRNA in a temporal fashion. Gene ontology analysis of mRNA m6A methylation content demonstrated the highest processes were consistent with m6A marking of mRNA for degradation or increased stability and indicated that modification of m6A mRNA methylation is an early cellular response to VEGFC stimulation. Correlating m6A mRNA methylation with mRNA abundance indicated that 83.2% of transcripts were significantly hypomethylated compared with only 3.6% hypermethylated. Calculating m6A stoichiometry for 12 hypomethylated transcripts relevant for hdLEC physiological processes by identification of all methylation sites in the 3’UTR to obtain percent methylation for unstimulated, 8-, and 24-hour VEGFC stimulation indicated that m6A methylation averaged 16.05% but dropped to 9.17 and 8.46% for 8- and 24-hour VEGFC stimulation, respectively. To test the hypothesis that reducing methylation would increase mRNA stability of these transcripts, we knocked down the m6A writer Mettl14 by transfection of hdLEC with specific siRNA in VEGFC-stimulated and Actinomycin D treated cells. The mRNA stability of transcripts which were hypomethylated upon VEGFC stimulation increased when Mettl14 was knocked down whereas hypermethylated transcript stability decreased. Importantly, in a lymphangiogenesis assay, hdLECs treated with Mettl14 siRNA formed significantly less tubes were significantly in response to VEGFC.
Together, this work indicates that VEGFC modifies m6A marking and mRNA stability of mRNA transcripts relevant for hdLEC pathophysiological processes and impacts lymphangiogenesis suggesting that methylation pathways may be leveraged as targets to impact lymphatic function.