Conclusion
Abovementioned results suggest that Nnat is the key to regulating the adipogenic differentiation of EASCs, and miR-3064-5p in EAT-derived exosomes can inhibit the expression of Nnat by targeting its mRNA, thereby affecting the adipogenic differentiation of EASCs.
Methods
EAT samples from CAHD patients and non-CAHD patients were collected to obtain exosomes via tissue culture. MiRNA sequencing was performed to analyze differences in miRNA expression in exosomes between groups. Luciferase reporter assay was then performed to verify the miRNA target gene. EAT was digested by collagenase to obtain EASCs, which were induced to mature adipocytes in vitro. Immunochemical staining and western blotting were performed to detect protein expression levels.
Results
The results showed that CAHD patients had higher levels of EASCs in EAT, and no significant difference in the adipogenic differentiation ability of EASCs was observed between CAHD and non-CAHD patients in vitro. This indicates that the EAT microenvironment is a key factor affecting the adipogenic differentiation of EASCs. The EAT-derived exosomes from CAHD patients inhibited adipogenic differentiation of EASCs in vitro. Sequencing analysis showed that miR-3064-5p was highly expressed in EAT-derived exosomes in CAHD patients, and its inhibitor could improve the adipogenic differentiation of EASCs. Luciferase reporter assay results showed that the target gene of miR-3064-5p is neuronatin (Nnat). Nnat remained silent in EASCs and was less expressed in EAT of CAHD patients.