| INTRODUCTIONPrimary aldosteronism (PA) is a common endocrine disorder characterized by autonomous hypersecretion of aldosterone and multi-system symptoms. The causes of primary aldosteronism determine the appropriate treatments and different results. Aldosterone-producing adenoma (APA) and bilateral idiopathic hyperaldosteronism (IHA) are the most common subtypes of PA. However, more than 90% of APA can be cured by adrenalectomy, but IHA often needs medical treatment. Although many authors made great progress in the pathology and prevention of PA, the detailed pathogenesis was still not very clear.Steroidogenic factor-1 (SF-1) is an important transcription factor. All steroidogenic synthases have one or more SF-1 binding sequences in the promoter region. SF-1 participates widely in regulating steroid synthesis, adrenal growth, tumorigenesis, and gonadal development. The terminal step in aldosterone biosynthesis is carried out by aldosterone synthase (CYP11B2), and angiotensin-Ⅱ(AT-Ⅱ) is the most important regulator of them. At present, the related studies on the role of SF-1 in aldosterone hormone secretion and PA pathogenitic mechanism were merely reported and the conclusion was not unified. In recent years, people have conducted the extensive research into CYP11B2 and APA, but most of them concentrated in case reports and individual target detection in tissues. However, there were rare documents reported in cell level. So we studied the role of SF-1 in the pathogenesis of PA through changing its gene expression. METHODS1. Sixteen APA samples were collected from patients with clinically and pathologically confirmed PA. Twelve normal adrenal samples came from patients who, due to renal carcinoma or renal pelvic carcinoma, underwent adrenalectomy along with renalectomy. SF-1 expression was determined by Western blotting and real-time polymerase chain reaction (PCR) method.2. Three DNA sequences of SF-1 which could transcripte small hairpin RNA(shRNA) were selected in GenBank database, and then clone into pGenesill plasmid. The most effective sequence, named pGenesill-SF-1-shRNA, was chosen by verification. The plasmid pGenesill-SF-1-shRNA was transfected into the H295R cells. SF-1 alteration was measured by Western blot and real-time polymerase chain reaction (RT-PCR).3. The H295R cell transfected pGenesill-SF-1-shRNA plasmid was consided as the experimental cell. RT-PCR and radioimmunoassay were used to detect CYP11B2 mRNA and aldosterone secretion levels in the control and experimental H295R cells respectively. CYP11B2 mRNA was determined by RT-PCR after AT-Ⅱstimulation at different time point, and the maximum gene expression level and peak hour could be determined. The sensitivities of CYP11B2 mRNA and aldosterone secretion to AT-Ⅱstimulation in the experimental and control cells were assessed by comparing the increases in CYP11B2 between 0 h and the peak hour.4. Cell proliferation was analyzed by WST-1 assay and cell count. Ki-67 expression was detected by immunohistochemistry and cell apoptosis was examined by TUNEL assay. All together, these were used to know the effects of SF-1 on H295R cell.RESULTS1. The SF-1 expression in APA was found to be higher than in normal adrenals regardless of mRNA or protein levels. A 10.48-fold increase in SF-1 mRNA was observed in APA in comparison to normal adrenals (P< 0.01). Moreover, the ratio of relative intensities in two groups was 0.87±0.05 and 0.39±0.07 (P< 0.01), respectively.2. The cloned shRNA was inserted into the pGenesil-1 vector and verified with enzyme digestion and DNA sequencing. The plasmid pGenesill-SF-1-shRNA was successfully gained. When specific SF-1-shRNA plasmid was used to transfect H295R cells, real-time PCR and Western blotting showed that SF-1 protein and mRNA levels in transfected cells were reduced by 69.7 and 71.2%(P< 0.01), respectively, compared with the control cells.3. Compared with the control cells, a 7.6-fold increase in CYP11B2 mRNA levels was observed in SF-1-shRNA-transfected cells under basal conditions. Similarly, the aldosterone production of SF-1-shRNA-transfected cells was approximately 2.04-fold higher than that of the control cells (0.112±0.010 vs.0.055±0.007 ng/ml, P=0.019). The highest CYP11B2 mRNA level was detected at the time of 12 hour after AT-Ⅱstimulation, and the amplification of SF-1- and control cells were 50.21 fold and 800.09 fold respectively comparing with those cells without AT-Ⅱstimulation (P<0.001). The latter was 15.93 times higher than the former (P<0.001).Similarly, Aldosterone production of control cells was (0.061±0.007) ng/ml and (0.256±0.014) ng/ml before and after AT-Ⅱintervention (P<0.01), and those of SF-1-transfected cells was (0.101±0.010) ng/ml and (0.252±0.016) ng/ml (P<0.01). The former amplification was 1.7 times higher than that of the latter(P<0.01).4. The control and experimental cells were cultured for four days, and cells numbers were counted on the last day. WST-1 and cell count method showed that SF-1 gene silencing obviously inhibited cell proliferation, the number of experimental and control cells were (6.25±0.25) x103 and (4.5±0.29) x103 at day 4, respectively (P<0.01). By contrast, there was a 3.7-fold increase in the percentage of apoptotic H295R cells in SF-1-inhibited group than that of control group (P<0.01). Immunohistochemistry showed that Ki-67 positive cells in SF-1-inhibited cells is lower than the negative control cells (16.9±2.17)% and (33.48±3.16)%, (P<0.01).CONCLUSIONS1. The up-regulation of SF-1 plays an important role in PA, and it may be become as a molecular target to study the pathogenesis of PA.2. SF-1 exerted a negative action on CYP11B2 in normal situation. Down-regulation of SF-1 decreases the sensitivity and response of CYP11B2 mRNA and aldosterone to AT-Ⅱstimulation after AT-Ⅱinterference. 3. SF-1 gene silencing can inhibit the proliferation of adrenocortical cells, and it is expected to become a key protein in understanding pathogenesis of adrenal tumors or treating them. |
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