miR-486-5p and miR-144-3p as candidate regulators of cortisol biosynthesis: functional and transcriptomic evidence in adrenocortical cells.

BACKGROUND

MicroRNAs (miRNAs) have been identified to regulate gene expression in adrenal disorders.

OBJECTIVE

To investigate the effects of specific miRNAs on cortisol biosynthesis.

METHODS

Seven candidate miRNAs were transfected as mimics into steroidogenically active adrenocortical cell lines (NCI-H295R, HAC15, and CU-ACC1). Steroids were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and ELISA. Quantitative polymerase chain reaction was performed in all cell lines with additionally microarray analysis in HAC15 cells. Adrenal RNA-sequencing data from patients with Cushing's syndrome (CS, n = 10) and normal controls (n = 8) were analyzed to compare differentially expressed genes (DEGs) and enrichments. miRNA inhibitors were further used for validation.

RESULTS

miR-486-5p and miR-144-3p mimics reduced cortisol levels by ∼60%-70% in NCI-H295R and HAC15 cells, and LC-MS/MS confirmed reduced steroidogenic metabolites. Both miR-486-5p and miR-144-3p mimics significantly inhibited CYP17A1 and CYP11B2 mRNA levels in NCI-H295R and HAC15 cells. Microarray analysis revealed the DEGs induced by miR-486-5p or miR-144-3p both predominantly enriched in "Metabolic Pathways," consistent with enrichment in the CS adrenal dataset. Common metabolic genes (co-DEGs) between patient samples and miRNA mimic-treated cells exhibited complementary expression patterns. Cotransfection of the corresponding inhibitors attenuated cortisol suppression and metabolic gene changes. Protein-protein interaction analysis further clustered the miR-486-5p- and miR-144-3p-associated co-DEGs into lipid metabolism-related biological process.

CONCLUSIONS

miR-486-5p and miR-144-3p suppress cortisol production in the human adrenocortical cell models of NCI-H295R and its derived HAC15 subclone and are associated with lipid metabolism-related networks, providing new insights into the molecular regulation of cortisol biosynthesis.