Circulating levels of mir-375 in cushing

Claudia Pivonello; Roberta Patalano; Tatiana Monto; Mariarosaria Negri; Feliciana Amatrudo; Donatella Provvisiero; Chiara Simeoli; Paola Nicola Di; Angelica Larocca; Erminio Crescenzo; Annamaria Colao; Rosario Pivonello

doi:10.1530/endoabs.99.P107

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Endocrine Abstracts (2024) 99 P107 | DOI: 10.1530/endoabs.99.P107

ECE2024 Poster Presentations Pituitary and Neuroendocrinology (120 abstracts)

Circulating levels of mir-375 in cushing’s disease patients and evaluation of its role in the regulation of sstr2 expression in murine pituitary corticotroph tumor cell model

Claudia Pivonello ¹ , Roberta Patalano ² , Tatiana Montò ² , Mariarosaria Negri ³ , Feliciana Amatrudo ³ , Donatella Provvisiero ² , Chiara Simeoli ² , Nicola Di Paola ² , Angelica Larocca ² , Erminio Crescenzo ² , Annamaria Colao ^2,4 & Rosario Pivonello ^2,4

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¹Università Federico II di Napoli, Dipartimento di Sanità Pubblica, Naples, Italy; ²Università Federico II di Napoli, Dipartimento di Medicina Clinica e Chirurgia, Sezione di Endocrinologia, Diabetologia. Andrologia e Nutrizione, Naples, Italy; ³Università Telematica Pegaso, Dipartimento di Scienze Umanistiche, Naples, Italy; ⁴Università Federico II di Napoli, UNESCO Chair for Health Education and Sustainable Development, Naples, Italy

In vitro studies suggest that glucocorticoids (GC) long-term exposure downregulates SSTR2 but not SSTR5 expression in human and mouse ACTH-secreting tumor cells (AtT20), limiting the efficacy of octreotide (OCT), a somatostatin-receptor ligand with high affinity for SSTR2, in the treatment of Cushing’s disease (CD). In AtT20, dexamethasone treatment increased miR-375 expression, whose analysis revealed a seed-sequence for SSTR2, supporting the hypothesis that excessive GC exposure can lead to epigenetic SSTR2 downregulation. The current study aims to evaluate miR-375 levels in CD patients and miR-375 impact on SSTR2 expression in AtT20/D16 cell model. Circulating and tissue miR-375 expression was evaluated by RT-qPCR in 21 CD patients and 19 healthy subjects; and in 6 human corticotroph pituitary tumors and 2 normal pituitaries, and in AtT20/D16 and somatotroph GH3 cell lines, respectively. SSTR2 protein expression and cellular localization were evaluated by western blot (WB) and immunofluorescence (IF) following miR-375 inhibition in AtT20/D16 in presence or not of OCT. Proliferation assay and flow cytometry were assessed to investigate the impact of miR-375 regulation on 72h of OCT at 10^-8M and 10^-7M treatment in AtT20/D16. Circulating miR-375 expression was higher in CD patients (P<0.0001) compared to healthy subjects, as well as tissue levels in human corticotroph pituitary tumors than in normal pituitaries. AtT20/D16 and GH3 cells exhibited an inverse pattern of expression, with low levels of SSTR2 messengers and high levels of miR-375 in AtT20/D16 and an opposite expression pattern in GH3 cells. miR-375 inhibition significantly increased membranous SSTR2 protein expression, evaluated by WB and IF, in AtT20/D16 (P=0.0310 and P=0.0154, respectively) compared to untreated cells. Receptor internalization, induced by 20 min of OCT10^-7M treatment, appeared stronger when OCT10^-7M was combined with miR-375 inhibitor. OCT10^-7M but not 10^-8M decreased cell proliferation (6.8%, P=0.011) compared to control. This effect was potentiated by miR-375 inhibition (miR-375inh+OCT10^-8M 9,0%, P=0.023 and miR-375inh+OCT10^-7M 10.1%, P=0.0001, vs control; miR-375inh+OCT10^-8M 8.5% P=0.0058 vs OCT10^-8M). Interestingly, miR-375 inhibition alone and in combination with OCT tend to increase the percentage of cells in early (miR-375inh 32.5%; miR-375inh+OCT10^-8M 25.7%; miR-375inh+OCT10^-7M 31.8%) and late apoptosis (miR-375inh 4.2%; miR-375inh+OCT10^-8M 4.9%; miR-375inh+OCT10^-7M 4.0%) compared to control (22.6% and 4.0%) and OCT alone (OCT10^-8M 31.8% and 3.2%; OCT10^-7M 27.8% and 2.5%) respectively, by inducing PARP, Caspase3 and ERK1/2 phosphorylation. Concluding, these data suggested that SSTR2 protein expression can be epigenetically downregulated by GC-induced increase of miR-375 expression at least partially and negatively influencing OCT action in corticotroph pituitary tumors.