Background
Endothelium-mesenchymal transition (EndMT) is a process of phenotypic and functional transition from activated endothelial cells to mesenchymal cells. Recently, EndMT has been proved to be one of the main pathological mechanisms of pulmonary artery hypertension (PAH). However, the molecular mechanism is not clear.
Conclusion
Our results elucidated that METTL3 knockdown inhibited the hypoxia-mediated EndMT process by inactivating TRPC6/calcineurin/NFAT signaling.
Methods
Primary rat pulmonary arterial endothelial cells (rPAECs) were isolated from Sprague-Dawley rats and verified by CD31 immunofluorescence staining. rPAECs were exposed to hypoxic conditions to induce EndMT. RNA and protein levels in cells were detected by RT-qPCR and Western blot. The migration ability was verified by the transwell assay. The RIP experiment was used to test the m6A modification of TRPC6 mRNA and the binding relationship between TRPC6 and METTL3. Calcineurin/NFAT signaling was measured by using commercial kits.
Results
METTL3 was found to be highly expressed by hypoxia treatment in a time-dependent manner. Knockdown of METTL3 significantly suppressed cell migration, downregulated the levels of interstitial cell-related markers like α-SMA and vimentin, and increased the levels of endothelial cell markers including CD31 and VE-cadherin. Mechanistically, METTL3 increased TRPC6 expression by enhancing the m6A modification of TRPC6 mRNA, thus activating calcineurin/NFAT signaling. Our experiments showed that METTL3 silencing mediated the inhibitory roles in the hypoxia-mediated EndMT process, which were significantly reversed by TRPC6/calcineurin/NFAT signaling activation.