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

Abstract

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 in vitro 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.

Potential Impact of N6-Methyladenosine RNA Methylation on Vision Function and the Pathological Processes of Ocular Diseases: New Discoveries and Future Perspectives

“…The expression of VEGFA and its splice variants, VEGFA-164 and VEGFA-188, is inhibited in METTL3-deficient bone marrow stromal cells [205]. In addition, METTL14, one of the m6A methylase components, plays an active role in regulating cardiovascular endothelial cell proliferation, invasion [206] and angiogenesis in haemorrhoids [207].…”

Research Progress for RNA Modifications in Physiological and Pathological Angiogenesis

“…Another in vivo experiment showed that silencing the eraser FTO in ECs can induce hyper-methylation on critical proangiogenic genes, such as FAK, which were recognized by the m 6 A reader YTHDF2 to induce RNA decay, thus regulating ocular angiogenesis (Shan et al, 2020). In comparison, angiogenesis is regulated by miR-4729, which inhibits METTL14 to decrease the m 6 A methylation of TIE1 and further inhibit TIE1/VEGFA signaling pathways in vascular ECs (Liu et al, 2021). In addition, FTO plays a vital role in cardiac contraction function, remodeling, regenerative repair, and cardiac homeostasis.…”

Insights into roles of METTL14 in tumors

“… 50 In vascular endothelial cells, the expression level of METTL14, modulated by miR‐4729, is reduced, resulting in declination in m6A modification level of TIE1 mRNA 3′UTR specific site, TIE1 mRNA stability reduction, and angiogenesis inhibition. 51 In renal cell carcinoma, METTL14 is regulated by the competitive binding effect of circRNAs and miRNAs, and affects the expression of downstream molecule PTEN and changes in related AKT/PKB signals. 52 In skin tumors, METTL14 acts as the target of NBR1‐dependent selective autophagy, and its expression level is down‐regulated.…”