METTL14 regulates M6A methylation-modified primary miR-19a to promote cardiovascular endothelial cell proliferation and invasion

B.-Y. Zhang, L. Han, Y.-F. Tang, G.-X. Zhang, X.-L. Fan, J.-J. Zhang, Q. Xue, Z.-Y. Xu

Department of Cardiovascular Surgery, Changhai Hospital of The Second Military Medical University, Shanghai, China. dr_xuzy@163.com

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• Cardiology

OBJECTIVE: Increasing evidence indicated that N6-methyl-adenosine (M6A) played a key role in a variety of pathophysiological processes. Methylases could promote the processing of mature mi-RNA in a M6A-dependent manner, thereby participating in the pathological cells’ occurrence and development. However, the regulatory mechanism of M6A in atherosclerosis (AS) was still unclear.

PATIENTS AND METHODS: Quantificational Real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of M6A, methyltransferase, demethylase transferase, miR-19a and other mi-RNA in atherosclerotic vascular endothelial cells (ASVEC). Cell Counting Kit (CCK8) was used to detect cell proliferation, the expression of PCNA was measured by Western Blot (WB) and qRT-PCR. Transwell assays were used to detect the invasion ability of ASVEC. Co-immunoprecipitation (Co-IP) was used to detect the binding of METTL14 to DGCR8. RNA Immunoprecipitation (RIP) was used to detect the binding of METTL14 to miR-19a.

RESULTS: M6A modification levels and METTL14 methylation transferase were significantly overexpressed in ASVEC. Silencing METTL14 inhibited the proliferation and invasion of ASVEC. Low expression of METTL14 suppressed the binding of methylated RNA and RNA splicing related protein DGCR8. Moreover, silencing METTL14 significantly inhibited the expression of miR-19a while promoted the expression of primary pre-miR-19a. However, high expression of METTL14 obviously increased the expression of DGCR8 and methylated m6A. Furthermore, silencing miR-19a inhibited the proliferation and invasion of ASVEC.

CONCLUSIONS: METTL14 increased the M6A modification of pri-miR-19a and promoted the processing of mature miR-19a, thus promoting the proliferation and invasion of ASVEC. These results suggested that METTL14/ M6A/ miR-19a signaling pathway may be a new target for atherosclerosis treatment.

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