MiR-4729 regulates TIE1 mRNA m6A modification and angiogenesis in hemorrhoids by targeting METTL14.

BACKGROUND

Hemorrhoids are a frequently-occurring disease of the anorectal system that is often accompanied by vascular hyperplasia and edema. A METTL14-mediated RNA N-6 methyladenosine (m6A) modification can improve mRNA stability and increase its transcriptional and translational activities, closely related to the occurrence of many diseases.

METHODS

Western blot, qPCR, and immunofluorescence staining were used to detect the levels of gene and protein expression. Haematoxylin and eosin staining was used for histopathological examination. RNA immunoprecipitation-PCR and RNA dot blotting were used to detect mRNA m6A modification.

RESULTS

Obvious signs of angiogenesis (CD31+/vWF+) were identified in the hemorrhoids. High levels of METTL14 expression on vascular endothelial cells (CD31+) suggested that angiogenesis was accompanied by differential modification of m6A RNA. It was subsequently found that the level of miR-4729 expression was significantly decreased in hemorrhoid tissues. The luciferase reporter enzyme assay results suggested that miR-4729 silenced its expression by targeting the 3'UTR of METTL14 mRNA. MiR-4729 overexpression in human umbilical vein endothelial cells (HUVECs) inhibited the proliferation and migration of HUVECs and vascular structure formation in the outer matrix. MiR-4729 overexpression significantly inhibited endogenous METTL14 expression in HUVECs and reduced the entire m6A RNA modification, especially the level of m6A methylation at the specific site of the 3' UTR of TIE1 mRNA. Moreover, miR-4729 overexpression significantly inhibited the molecular loop of the TIE1/VEGFA signaling pathway in HUVECs.

CONCLUSIONS

Our findings confirmed that the down-regulation of miR-4729 in hemorrhoid vascular endothelial cells was one of the main reasons for vascular proliferation. The overexpression of miR-4729 in vascular endothelial cells decreased the global mRNA methylation and TIE1 mRNA 3'UTR-specific site methylation by silencing METTL14 expression, reducing TIE1 mRNA stability, down-regulating the TIE1/VEGFA signal molecular loop expression, and weakening angiogenesis ability.