Abstract Body: Therapeutic targeting of non-coding RNAs, particularly microRNA (miRNA), presents an appealing approach in the treatment of chronic diseases such as resistant hypertension. Recently, miRNA have emerged as promising targets for therapeutic intervention, capable of regulating critical genes to provide a suitable solution to the lack of efficacy of current treatments. Our comprehensive miRNA profiling of plasma extracellular vesicles (EV) from DOCA-salt hypertensive mice of both sexes, compared to sham controls, identified two miRNA implicated in salt-sensitive hypertension, namely miR-125b-5p and miR-125a-5p. Our bioinformatic analysis suggests that these miRNA could be involved in angiotensin-converting enzyme 2 (ACE2) downregulation in hypertension. The present study intended to validate ACE2 as a target of miR-125. Quantitative RT-PCR and capillary western analysis of hypothalami from C57BL/6J DOCA+Salt hypertensive male and female mice following 3-weeks of DOCA+Salt treatment (n=5-6 per group, 3-month old), detected a significant decrease in ACE2 mRNA (males: 0.34±0.03 vs. 1.02±0.02; females: 0.65±0.08 vs. 1.01±0.01; P<0.05) and protein (males: 0.17±0.02 vs. 0.31±0.05; females: 0.16±0.03 vs. 0.37±0.07; P<0.05) expression for both sexes. Further investigation in bEND.3 cells treated with different concentrations of Ang-II (0, 10, 100 nM) for 12, 24, and 48 hours, demonstrated a dose-dependent negative correlation between miR-125b-5p (r≈-0.9978) or miR-125a-5p (r≈-1.002) and ACE2 gene expression, independently of time. When treated with miR-125 mimics the cells demonstrated a consistent downregulation of ACE2 protein levels (miR-125b-5p: 0.6±0.1 and miR-125a-5p: 2.0±0.2 vs. control: 2.8±0.3). Conversely, no significant change in ACE2 protein levels was observed with the respective miRNA inhibitors. Using a firefly/Renilla luciferase assay, we further identified ACE2 as a validated primary target for these miRNA. While miR-125b-5p indirectly modulates the expression of the Ace2 gene by targeting important transcription factors like Sp1 and STAT3, our data further elucidate that miR-125b-5p and/or miR-125a-5p directly reduce ACE2 levels by targeting ACE2 3’UTR mRNA. Additional work is warranted to investigate these specific regulatory mechanisms based on cell specificity, physiological conditions, and sex differences.