Regulatory effect of steroidogenic factor-1 gene silencing on aldosterone and cortisol secretion to adreno-cortico-tropic-hormone stimulation in H295R cells during acute and chronic phases

Abstract

Objective To study the changes in aldosterone and cortisol secretion in human adrenocortical carcinoma H295R cells after adreno-cortico-tropic-hormone (ACTH) stimulation,and investigate the effect of steroidogenic factor-1 (SF-1) gene silencing on them.Methods Lentiviral vector pLVX-specific short hairpin RNA (shRNA)-SF-1 containing SF-1-specific shRNA was transfected into the H295R cells,and negative-control and black groups were set up simuhaneously.SF-1 expression was detected by Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) at 48 h after transfection.Steroidogenic acute regulatory protein (StAR) mRNA was detected at 1 h after ACTH stimulation (100 nmol/L),and CYB1 1 B2/B1 mRNA was detected at 48 h after ACTH stimulation (100 nmol/L).Aldosterone and cortisol were measured by enzyme linked immunosorbent assay (ELISA) analysis,and the increase amplitude was compared.Results As compared with those in control group,the protein and mRNA levels in pLVX-shRNA-SF-1-transfected cells were reduced by 69％ and 71％ respectively.StAR mRNA in transfection group was 1.4-fold higher than that of the control group at 1 h after ACTH stimulation (P ＜ 0.05).Similarly,the aldosterone production in two groups was (91.59-± 4.28) and (79.90 ± 2.41) ng/L (P＜0.05),and cortisol production was (23.72 ± 1.50) and (23.74 ± 1.40) ng/L (P＞0.05),respectively.A 13-fold increase in CYP1 1 B2 mRNA levels was observed in transfection group as compared with the control group at 48 h after ACTH stimulation (P ＜0.05).However,aldosterone levels in two groups were (129.50±6.87) and (131.52 ± 7.56) ng/L respectively (P ＞ 0.05).CYP11B1 mRNA was 1.18-fold higher in transfection group than that of the control group (P ＞ 0.05),and cortisol production was (28.95 ± 1.51) and (33.08 ± 1.67) ng/L respectively (P ＜ 0.05).For aldosterone and cortisol,the amplification in control group was 1.37 and 1.80 times higher than that of transfection group (P ＜ 0.05).Conclusion Down-regulation of SF-1 decreases the sensitivity and response of aldosterone and cortisol to ACTH stimulation. Key words: Steroidogenic factor-1 ; Adrenocortical carcinoma; adreno-cortico-tropic-hormone; Aldosterone; Cortisol