Research Paper Volume 11, Issue 14 pp 5232—5245

Aberrant DNA methylation of synaptophysin is involved in adrenal cortisol-producing adenoma

Figure 4. TET3 is a direct target of miR-27a-5p and TET3 regulated SYP expression and cortisol secretion in H295R cells. (A) RNA22 predicts that TET3 is a potential target of miR-27a-5p. (B) Immunohistochemical staining for TET3 in CPA and normal adrenal tissue. DAB staining showed that the intensity of staining for TET3 was significantly stronger in CPAs than in normal adrenal tissues. The brown area indicated by arrow is the positive staining of TET3 in CPAs. Representative data are shown. (C) TET3 protein levels in H295R cells were assessed by Western blotsin control in miR-27a-5p mimics transfected cells and miR-27a-5p inhibitor transfected cells. (D) Luciferase reporter assays were performed using luciferase constructs carrying a WT or mutant TET3 3_-UTR cotransfected into H295R cells with miR-27a-5p mimics compared with an empty vector control. Firefly luciferase activity was normalized to renilla luciferase activity. (E) Western blot analysis of protein levels in H295R cells transfected with TET3 siRNA (siRNA 1#,2#) or scrambled control for 48h. (F) Methylation level of SYP promoter was analysis by EpiTect Methyl II PCR Assay in H295R cell transfected with TET3 siRNA or control. (G) Cortisol secretion were measured in cell supernatants by ELISA from H295R cell transfected with TET3 siRNA or control. Error bars represent SD. Three independent experiments were performed, and representative data are shown. The data represent the mean SD. NC, normal control. **p<0.01. *p<0.05.