MiR-375 Negatively Regulates The Synthesis And Secretion Of Catecholamines By Targeting Transcription Factor Sp1 In Rat Adrenal Medulla

Adrenal gland is an important endocrine gland synthesizing and secreting catecholamines (CATs)which play crucial roles in balancing homeostasis. It has been documented that miRNAs, including miR-375, play key roles in regulating the synthesis and secretion of many endocrine hormones, such as insulin and estrogen. However, up to now, there are few studies involving in the regulation mechanism of miRNAs on the synthesis and secretion of CATs. Our recent studies have shown that miR-375 is highly expressed in the adrenal gland, but the related biological functions still remain unclear. The aims of the present study were to investigate the functions and related mechanisms of miR-375 in the adrenal gland.In this study, we initially detected miR-375 expression in different tissues of adult rat by real-time quantitative PCR (qPCR). The results showed that miR-375 was highly expressed in the adrenal gland.Its expression level gradually increased during the development of the adrenal gland and reached the highest level in adult. In addition, the in situ hybridization (ISH) results showed that miR-375 specifically expressed in the adrenal medulla cells and no signal was detected in the cortex.In order to identify the functions of miR-375 in the adrenal gland, we used the immobilization stress (IMO) rat model to study the functions of miR-375 in response to stress. The results showed that the miR-375 expression in the adrenal gland was down-regulated but the CATs levels in rat plasma were increased under stress. Further, we assayed the functions of miR-375 in adrenal medulla cells by inhibiting and overexpressing miR-375 in cultured primary adrenal medulla cells and PC 12 cells using miR-375-inhibitor (miR-375-in) and miR-375-mimics (miR-375-mi). The results demonstrated that miR-375-in increased the CATs levels in the culture medium, whereas miR-375-mi decreased the CATs levels in the culture medium. In addition, our results showed that the inhibition of endogenous miR-375 significantly enhanced tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) expressions,while miR-375 overexpression sharply down-regulated TH and DBH expressions in primary adrenal medulla cells and PC 12 cells. However, miR-375 did not participate in the degradation of CATs and the negative feedback process of norepinephrine (NE) to CATs. Moreover, we confirmed that miR-375 had no significant effect on proliferation and apoptosis of PC 12 cells by BrdU cell immunochemistry,TUNEL and qPCR. These results indicate that miR-375 negatively regulates the CATs synthesis and secretion.Bioinformatics was applied to predict the target gene of miR-375, and Specific protein 1 (Spl) was selected as a putative target gene of miR-375. The results from luciferase rqorter assay and western blot analysis showed that Spl was a direct target gene of miR-375. Meanwhile, Sp1 siRNA interference and Spl overexpression experiments proved that Spl involved in the miR-375 mediated signaling pathway of TH and DBH expressions. In addition, Sp1 protein expression increased under the IMO stress. It was double-checked that Spl was the target gene of miR-375 and participated in the stress response.In conclusion, our results demonstrate that miR-375 is specifically expressed in the rat adrenal medullary chromaffin cells and plays as a negative regulator of CATs biosynthesis and secretion in the response to stress. In light of our results, miR-375 negatively regulates the expressions of TH and DBH via transcription factor Spl and further influences the synthesis and secretion of CATs in the adrenal gland. These results provide a new basis for the study of miRNAs in CATs synthesis and secretion.