PS-B04-9: ELUCIDATING EFFECT OF INHIBITING ALDOSTERONE SYNTHESIS ON HUMAN ADRENOCORTICAL CELLS

Abstract

Introduction: Modern human diet is high in salt. High salt stimulates decrease of aldosterone by down-regulation of aldosterone synthase (encoded by the gene CYP11B2) in the zona glomerulosa (ZG). Aldosterone regulates blood pressure and volume by regulating salt secretion. Interestingly, adult human adrenal ZG is sparse and confined to discrete islets of cells unlike rodent adrenal where the zone encompasses the whole periphery of the adrenal. We therefore hypothesize that ZG cell function change from synthesis to apoptosis/migration mode when aldosterone is not required due to the high salt diet. Objective: Herein, we intend to characterise human adrenocortical cells silenced for CYP11B2. Methods: We transfected HAC15, a subclone of the H295R human adrenocortical cancer cell line with scrambled siRNA (siScrambled) or siRNA targeting CYP11B2 (siCYP11B2) using the Neon Transfection System. 48-h post-transfection, cell apoptosis rate was assessed using the Pacific Blue Annexin V/SYTOX AADvanced apoptosis kit. The supernatants and cells were harvested for aldosterone and cortisol measurements, and RNA isolation. A representative sample of siCYP11B2 and siScrambled from each independent experiment was selected for RNA-Sequencing (RNAseq). The EZMTT Proliferation Assay was performed on equally seeded HAC15 cells, 72-h post-transfection with either siScrambled or siCYP11B2 using the Lipofectamin 3000 Transfection reagent. Experiments were repeated 3 times independently in triplicates. Results: siCYP11B2 transfection limited the production of aldosterone by 69.8% (p = 0.001) and down-regulated the expression of CYP11B2 by 83.0% (p = 0.001). We confirmed that the silencing was specific to CYP11B2 despite the gene being highly homologous to CYP11B1 as there were no significant change on cortisol production and CYP11B1 mRNA expression. RNAseq analysis found 3 genes, CYP11B2, HNRNPA1, and UHMK1, consistently down-regulated in siCYP11B2 by 32%, 65% and 67% respectively. The most enriched pathways among the differentially expressed genes (DEG) were mitophagy and autophagy. No difference in cell apoptosis was seen in siCYP11B2 cells. Instead, an increase in cell proliferation was detected with the EZMTT assay. Conclusion: Our findings suggest that silencing of CYP11B2 does not affect apoptosis but does affect cell proliferation. Interestingly, the mitophagy and autophagy pathways were enriched in the DEGs of siCYP11B2 cells.