miR-493-5p Silenced by DNA Methylation Promotes Angiogenesis via Exosomes and VEGF-A-Mediated Intracellular Cross-Talk Between ESCC Cells and HUVECs.

ABSTRACT

Background: Exosomal microRNAs (miRNAs) in the tumor microenvironment play crucial roles in tumorigenesis and tumor progression by participating in intercellular cross-talk. However, the functions of exosomal miRNAs and the mechanisms by which they regulate esophageal squamous cell carcinoma (ESCC) progression are unclear. Methods: RNA sequencing and GEO analysis were conducted to identify candidate exosomal miRNAs involved in ESCC development. Receiver operating characteristic curve analysis was performed to assess the diagnostic value of plasma exosomal miR-493-5p. EdU, tube formation and Transwell assays were used to investigate the effects of exosomal miR-493-5p on human umbilical vein endothelial cells (HUVECs). A subcutaneous xenograft model was used to evaluate the antitumor effects of miR-493-5p and decitabine (a DNA methyltransferase inhibitor). The relationship between miR-493-5p and SP1/SP3 was revealed via a dual-luciferase reporter assay. A series of rescue assays were subsequently performed to investigate whether SP1/SP3 participate in exosomal miR-493-5p-mediated ESCC angiogenesis. Results: We found that miR-493-5p expression was notably reduced in the plasma exosomes of ESCC patients, which showed the high potential value in early ESCC diagnosis. Additionally, miR-493-5p, as a candidate tumor suppressor, inhibited the proliferation, migration and tube formation of HUVECs by suppressing the expression of VEGFA and exerted its angiostatic effect via exosomes. Moreover, we found that SP1/SP3 are direct targets of miR-493-5p and that re-expression of SP1/SP3 could reverse the inhibitory effects of miR-493-5p. Further investigation revealed that miR-493-5p expression could be regulated by DNA methyltransferase 3A (DNMT3A) and DNMT3B, and either miR-493-5p overexpression or restoration of miR-493-5p expression with decitabine increased the antitumor effects of bevacizumab. Conclusion: Exosomal miR-493-5p is a highly valuable ESCC diagnosis marker and inhibits ESCC-associated angiogenesis. miR-493-5p can be silenced via DNA methylation, and restoration of miR-493-5p expression with decitabine increases the antitumor effects of bevacizumab, suggesting its potential as a therapeutic target for ESCC treatment.