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Endocrine Abstracts (2017) 49 OC5.1 | DOI: 10.1530/endoabs.49.OC5.1

1Pontificia Universidad Católica de Chile, Santiago, Chile; 2Universidad del Desarrollo, Santiago, Chile; 3University of Queensland, Brisbane, Australia.


Arterial hypertension is a major health problem affecting 1.13 billion people worldwide, of which 10% could be due to endocrine pathologies related to the mineralocorticoid receptor (MR) (i.e. primary aldosteronism, 11β-hydroxysteroid dehydrogenase type 2 enzyme (HSD11B2) deficiency). The MR pathway is expressed mainly in placenta, kidney and colon epithelial cells. Current research highlights potential epigenetic regulatory mechanisms for the MR pathway. Here, specific miRNAs are being studied as epigenetic regulators, either present in cells or exosomes (nanovesicles of 40–150 nm diameter) carrying miRNA, RNA, lipids and proteins. Those miRNAs could dynamically influence the onset and progression of the mineralocorticoid arterial hypertension (AH).

Aim: To isolate exosomes from polarized epithelial cells and to identify in their cargo miRNAs affecting HSD11B2 and MR gene expression.

Methods: The bioinformatic tool mirWalk2.0 was used to determine miRNAs capable to interact with HSD11B2 and MR RNAs. The cell line derived from colon epithelia (Caco-2) cells was cultured and polarized in Transwells. MR pathway genes and miRNAs targeting the MR and HDS11B2 were measured by qPCR. Exosomes from apical and basolateral culture medium were isolated at 24 h of incubation in basal conditions, quantified with NanoSight-NS300 and their specific miRNA content was determined.

Results: We identified bioinformatically that miRNA 135b, 644a and miRNA 488, 1205 could interact with the MR and HSD11B2, respectively. Caco-2 cells polarize after 21 days of culture (trans-epithelial resistance (TEER): 114.18 cm2), expressing polarization genes (SLC11A2, SLGT1, ALPI), genes associated to MR pathway (NR3C2, HSD11B2, SCNN1A, SLC12A2, SGK1, ATP1A1) and pre-miRNAs (135b, 644a, 488, 1205). We identified that Caco-2 release 21×109±0.69×109 (apical) and 4.1×109±0.0029×109 (basolateral) extracellular vesicles per ml of which a 36.8 and 38.5% are exosomes with a modal range in 124–125 nm diameter respectively. Preliminary results show expression of miRNA 135b, 644a, 488 and 1205 in apical and basolateral exosomes.

Conclusion: Caco-2 cells release five-times more exosomes in the apical than basolateral side, both carrying miRNAs that could interact HSD11B2 and MR gene.

Volume 49

19th European Congress of Endocrinology

Lisbon, Portugal
20 May 2017 - 23 May 2017

European Society of Endocrinology 

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