MiR‐193a‐3p functions as a tumour suppressor in human aldosterone‐producing adrenocortical adenoma by down‐regulating CYP11B2

Abstract

The mechanism of aldosterone-producing adrenocortical adenoma (APA) pathogenesis and the role of microRNAs (miRNAs) in APA pathogenesis have not been completely clarified. We examined the expression and function of miR-140-3p, miR-193a-3p and miR-22-3p, which have binding sites in CYP11B2. Expression of miRNAs and CYP11B2 mRNA was measured by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation was monitored by colorimetric analysis, and cell apoptosis and cell cycle progression were analysed by flow cytometry. ELISA was carried out to detect aldosterone levels in cell culture supernatants. Luciferase reporter assays, qRT-PCR and Western blotting were performed to identify CYP11B2 as a target of miR-193a-3p. Of the three miRNAs examined, miR-193a-3p exhibited a significant decrease and CYP11B2 mRNA exhibited a significant increase in expression in APA compared with adjacent normal adrenal gland tissue. Transfection of miR-193a-3p mimic into the human adrenocortical cell line H295R showed that elevated miR-193a-3p expression inhibits proliferation and aldosterone secretion, induces G1-phase arrest and promotes apoptosis in H295R cells. Furthermore, in luciferase reporter assays, overexpression of miR-193a-3p in H295R cells significantly reduced the luciferase activity of the wild-type CYP11B2 3'-UTR construct, which could be reversed by mutation of the miR-193a-3p-binding site. Moreover, miR-193a-3p overexpression downregulated CYP11B2 mRNA and protein expression. Finally, overexpression of CYP11B2 diminished the effects of miR-193a-3p on H295R cells. Taken together, our results suggest that CYP11B2 levels may be modulated by miR-193a-3p in APA, which could explain, at least partially, why downregulation of miR-193a-3p during APA formation may promote cell growth and suppress apoptosis.

MicroRNAs and Adrenocortical Tumors: Where do we Stand on Primary Aldosteronism?

“…The role of hsa-miR-193a-3p has been previously reported in the regulation of cell proliferation and apoptosis in ovarian cancer cells and non-small cell lung cancer, where hsa-miR-193a-3p was found to induce apoptosis and suppress tumor cell proliferation [37,[51][52][53]. Furthermore, hsa-miR-193a-3p was found to suppress cell proliferation in human endothelial cells [37,54]. The underexpression of hsa-miR-193a-3p in ovarian cancer cells and non-small cell lung cancer as a tumor suppressor miRNA is in concordance with the findings of Zhang et al, reporting hsa-miR-193a-3p as the most downregulated miRNA in APAs [37].…”

“…There are only few studies using high-throughput methods to identify miRNA expression alterations in APAs. The potential role of miRNAs as direct epigenetic regulators of CYP11B2 has also been proposed by predicting several miRNA-binding sites in the 3' UTR of CYP11B2 [37].…”

“…These findings proposed a possible pathogenic role of hsa-miR-193a-3p in APAs via suppression of cell proliferation and promotion of apoptosis. In the NCI-H295R cell line, overexpression of hsa-miR-193a-3p resulted in a significant inhibition of cell proliferation via the induction of G1-phase arrest, cell apoptosis and inhibition of aldosterone secretion, and thus it might be a major factor in the pathogenesis of APA [37]. Furthermore, the direct regulatory effect of hsa-miR-193a-3p on its possible target mRNA CYP11B2 was also investigated by in vitro luciferase reporter assay [37].…”

“…Furthermore, the direct regulatory effect of hsa-miR-193a-3p on its possible target mRNA CYP11B2 was also investigated by in vitro luciferase reporter assay [37]. Hsa-miR-193a-3p was confirmed to bind the CYP11B2 mRNA [37]. By overexpressing CYP11B2 in NCI-H295R cells, the antiproliferative and proapoptotic effects of overexpressed hsa-miR-193-a-3p could be attenuated, and therefore it was hypothesized, that the primary action of hsa-miR-193a-3p could be its inhibition on CYP11B2 [37].…”

Investigating the Role of Mineralocorticoid Receptor Signaling in Cancer Biology in the Genomic Era

“…Somatic mutations occurring in KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes give rise to sporadic APA [61]. In addition to the abovementioned mutations, regulatory RNAs such as miRNAs have also been shown to be important in modulating aldosterone levels and tumorigenesis [62][63][64]. On the other hand, adrenocortical cancers (ACC), some of which are hormone-producing, occur relatively rarely, and patients with ACC exhibit poor prognosis with a median survival of 5.5 years [65].…”