Influences of steroidogenic factor-1 gene silencing on angiotensin II -mediated aldosterone secretion

Abstract

Objective To observe the changes of CYP11B2 mRNA and aldosterone secretion in human adrenocortical carcinoma H295R cells after angiotensin Ⅱ (AT Ⅱ ) stimulation, and investigate the effects of steroidogenic factor-1 ( SF-1 ) gene silencing. Methods The plasmids pGenesil1-SF-1-shRNA containing U6 promoter and SF-1 -specific short hairpin RNA (shRNA) or pGenesil1 -negative-shRNA containing unspecific shRNA were transfected into the H295R cells. The expression of SF-1 was detected by Western blotting and real-time polymerase chain reaction (RT-PCR). CYP11B2 mRNA was detected at 3,6, 9, 12, 15, 18 h after ATⅡ stimulation ( 100 nmol/L), and the maximal amplitude was compared between two groups. Aldosterone was measured by radioimmunoassay at 3, 6, 9, 12, 15, 18 h after AT Ⅱ stimulation ( 100 nmol/L), and the amplitude of aldosterone was compared between two groups. Results As compared with those in control group, the protein and mRNA levels in SF-1-transfected group were reduced by 69.07% and 71.20% respectively. The highest CYP11B2 mRNA level was detected at 12 h after ATⅡ stimulation, and the amplitude in SF-1-transfected group and non-transfection group was 50. 21 fold and 800.09 fold respectively compared to that of cells without ATⅡ stimulation ( P ＜ 0.01 ). Similarly, aldosterone secretion in control group was (0. 061 ± 0. 007 ) and (0. 256 ± 0.014) μg/L before and after ATⅡ intervention (P＜0. 01), and that in SF-1-transfected group was (0. 101 ±0.010) and (0.252 ±0.016) μg/L (P ＜ 0. 01 ) respectively. Conclusion Down-regulation of SF-1 decreased the sensitivity and response of CYP11B2 mRNA and aldosterone to ATⅡ stimulation. Key words: Steroidogenic factor-1; H295R cell; AngiotensinⅡ; Aldosterone