Steroid Factor-1 And Dax-1 Adrenal Cortical Tumor

Objectives:Adrenal masses can be detected in up to 4%of the population,and are mostly of adrenocortical origin,in the last decade,studies of the genetics of Adrenocortical tumouts (ACTs) have led to major advances in this field.The identification of germline molecular defects in the hereditary syndrome responsible for ACTs has facilitated progress in sporadic tumours.Steroidogenic factor-1(SF-1)and Dosage-sensitive sex reversal,adrenal hypoplasia congenital,critical region on the X chromosome gene-1(DAX-1 )are two orphan nuclear receptors that have demonstrated to contribute significantly to adrenocortical function.Studies have also shown that SF-1 overexpressin can cause adrenocortical nodule and tumor formation in mice,meanwhile,DAX-1 might inhibit the proliferation of endometrial carcinoma.It is suggested that SF-1 and DAX-1 might also be critical for adrenal tumorigenesis.The aim of this study is to detect SF-1,and DAX-1 mRNA expression in human adrenal tumors and normal adrenal cortex so as to gain an insight into the action of SF-1 and DAX-1 on the steroidogenesis and development of adrenocortical tumors.Methods:1.Total RNA was extracted from 17 adrenal aldoterone-producing adenomas(APA),include 12 angiotensin-unresponsive aldosterone -producing adenom(AⅡ-U-APA) and 5 angiotensin-responsive aldosterone-producing adenoma(AⅡ-R-APA),10 cortisol-producing adenomas(CPA),10 adrenal non-functional adenomas(NFA),2 aldosterone-secreting adrenocortical carcinomas(ACC),6 pheochromocytomas and 8 normal human adrenal cortex,and then the total RNA was reverse transcripted to cDNA.2.Real-time quantitative PCR was carried out to analyze gene expression of SF-1, DAX-1 and ACTH receptor in different tissues.The expression levels of the target genes were relatively quantified usingβ-actin as internal control.3.The expression levels of the target genes were compared in diffent tissues.It was studied whether the mRNA expression of ACTH receptor,SF-1 and DAX-1 correlated with clinical data in adrenocortical tumors.Results:1.The mRNA expression of SF-1 in adrenocortical tumors1 ) No significant difference was found between APA and normal adrenal cortex.SF-1/β-actin was 23.89±:3.17 in AⅡ-U,21.59±3.00 in AⅡ-R-APA and 24.58±2.45 in normal adrenal cortex.2 ) The expressin in CPA(14.17±2.80)was much lower than normal adrenal cortex and APA(all p<0.01).3) The expressin in NFA(36.38±3.50) was much higher than normal adrenal cortex,APA and CPA(all p <0.01).4) SF-1/β-actin in the two aldosterone-producing carcinoma(38.75 and 44.16)tended to be higher than other tissues.2.The mRNA expression of DAX-1 in adrenocortieal tumors1) Significant different was found between APA and normal adrenal cortex.DAX-1/β-actin was 0.37±0.05 in AⅡ-U-APA(p<0.01),0.43±0.05 in AⅡ-R-APA(p<0.05) and 0.57±0.06 in normal adrenal cortex2) It was much lower in CPA(0.39±0.04) than normal adrenal cortex(p< 0.01).3 ) It was much higher in NFA(0.83±0.08) than normal adrenal cortex,APA and CPA(all p <0.01).4) DAX-1/β-actin in the two aldosterone-producing carcinoma(1.51 and 1.21)tended to be higher than other tissues.3.The mRNA expression of ACTH receptor in adrenoeortical tumors1 ) The ACTH receptor/β-actin in AⅡ-U-APA(1.41±0.18) was higher than AⅡ-R-APA(1.17±0.25 )(p<0.05).It was much higher in the two groups than normal adrenal cortex(0.94±0.10)(p<0.01 or 0.05) 2) It was much lower in CPA(0.55±0.05) than APA and normal adrenal cortex.3) It was also much lower in NFA(0.71±0.26) than APA and normal adrenal cortex.(p<0.05 or 0.01).4) ACTH-receptor/β-actin in the two aldosterone-producing carcinoma(0.48 and 0.43)tended to be lower than other tissues.4.The ratio of SF-1 to DAX-1 in adrenocortical tumors1) The SF-1/DAX-1 ratio in AⅡ-U-APA(58.78±12.08) was higher than normal adrenal cortex(43.70±6.44),AⅡ-R-APA(46.19±5.64 ),CPA(36.16±6.89) and NFA(44.00±5.32)(p<0.05 or 0.01).2) The ratio in AⅡ-R-APA was also higher than CPA(p<0.05).5.The correlation of ACTH receptor,SF-1 and DAX-1 mRNA expression with the clinical data in adrenocortical tumors1 ) Aldosterone-producing adenomaa).In AⅡ-U-APA,the increase of plasma aldosterone level to upright stimulation had a negative correlation with the ACTH receptor mRNA expression(r=—0.663,p = 0.019),and had positive correlation with DAX-1mRNA(r=0.655,p =0.021).I It also had negative correlation with SF-1/DAX-1 ratio(r= -0.432,p=0.046)b).SF-1/DAX-1 ratio had a negative correlation with tumor size(r=-0.712,p = 0.009)(all p <0.05 )c).In AⅡ-R-APA,the increase of plasma aldosterone level to upright stimulation only had a positive correlation with the ACTH receptor mRNA expression(r =0.900,p=0.037),and had negative correlation with DAX-1mRNA(r= -1.000,p=0.000).2) Cortisol-producing adenomaa).The ACTH receptor,SF-1 and DAX-1 mRNA expression in CPA had no correlation with plasma and urine cortisol levels.b).The tumor size only had positive correlation with SF-1mRNA(r=0.695,p =0.026).6.The mRNA expression of SF-1,DAX-1 and ACTH receptor was not detected in pheochromocytoma tissues.Conclusions:1.The mRNA expression of SF-1 and DAX-1 was different in APA,CPA,NFA and aldosterone-producing carcinoma,which indicated that they might have tight correlation with adrenal tumorigenesis.2.The normal mRNA expression of SF-1,lower expression of DAX-1,higher SF-1/DAX-1 ratio in AⅡ-U-APA tissues and negative correlation of the ratio with tumor size is suggested that low DAX-1 might affect the development of AⅡ-U-APA.3.SF-1 and DAX-1mRNA tended to be overexpressed in NFA and aldosterone-secreting adrenocortical carcinoma,it is suggested that they could accelerate the tumorigenesis of aldosterone-secreting adrenocortical carcinoma,and we should also follow the prognosis of NFA patients.4.The lower expression of SF-1mRNA in CPA had positive correlation with tumor size,which indicated than SF-1 might have tight correlation with tumorigenesis.5.The mRNA expression of SF-1 and DAX-1 was not detected in pheochromocytoma tissues indicated that they might have nothing to do with the development of pheochromocytoma.6.Some outcomes had not been reported as yet. Objectives:The purpose of this study is to detect the protein level of steroidogenic factor-1(SF-1) and DAX-1 in human adrenal aldosterone-producing adenoma,cortisol-producing adenoma,non-functional adrenocortical tumor,adrenocortical carcinoma and normal adrenal cortex,to investigate the roles of SF-1 and DAX- 1 in the steroidogenesis and development of adrenocortical tumors especially of aldosterone-producing adenoma.Methods:1.Total protein was extracted from 17 adrenal aldoterone-producing adenomas,,include 12 angiotensin-unresponsive aldosterone -producing adenom (AⅡ-U-APA) and 5 angiotensin-responsive aldosterone-producing adenoma(AⅡ-R-APA),10 cortisol-producing adenomas(CPA),8 adrenal non-functional adenomas(NFA),2 aldosterone-secreting adrenocortical carcinomas(ACC) and 8 normal human adrenal cortex,and then The concentration of protein was evaluated by the means of Bradford.2.Using western blot we analyzed the protein expression of SF-1,DAX-1 andβ-actin.The protein expression of SF-1,DAX-1 was semi-quantified by dividing the grayness value of SF-1/β- actin and DAX-1/β- actin in the same sample and compared among the different tissues.3.It was studied whether the protein expression of SF-1 and DAX-1 correlated with clinical data in adrenocortical tumors,and the difference between mRNA and protein expression of SF-1 and DAX-1 were also compared.Results:1.The protein expression of SF-1 in adrenocortical tumors1 ) It was 0.76±0.10 in AⅡ-U-APA,0.73±0.07 in AⅡ-R-APA.And 0.76±0.11 in normal adrenal cortex,No significant difference was found between the three groups(p>0.05 ).2) SF-1 protein was much lower in CPA(0.55±0.04) than normal adrenal cortex and APA(all p<0.01).3 ) It was much higher in NFA(0.98±0.10) than normal adrenal cortex,APA and CPA(all p <0.01).4) SF-1 protein tended to be higher in adrenocortical carcinoma(1.24 and 1.40) than other tissues.2.The protein expression of DAX-1 in adrenocortical tumors1) AⅡ-U-APA(0.39±0.13) and AⅡ-R-APA(0.43±0.14) was much lower than normal adrenal cortex(0.65±0.14)(p <0.01).2) In CPA(0.56±0.04),it was lower than normal adrenal cortex and higher than AⅡ-U-APA(p <0.05 or 0.01).3 ).It was higher in NFA(1.03±0.13) than other tissues(all p <0.01).4) DAX-1 protein tended to be higher in adrenocortical carcinoma(1.18 and 1.02) than other tissues.3.The SF-1/DAX-1 protein ratio in adrenocortical tumors1 ) The ratio in AⅡ-U-APA(2.28±0.79) and AⅡ-R-APA(1.56±0.44) was higher than other tissues(all p<0.01 ),2) The ratio in AⅡ-U-APA was higher than AⅡ-R-APA(p <0.01 ).4.The correlation of SF-1 and DAX-1 protein expression with the clinical data in adrenocortical tumors1 ) The increase of plasma aldosterone level to upright stimulation in AⅡ-U-APA had positive correlation with DAX-1 protein(r=0.616,p=0.033) and had negative correlation with SF-1/DAX-1 ratio(r=-0.588,p=0.044).2) SF-1/DAX-1 ratio also had a negative correlation with tumor size(r=-0.580, p =0.048).3) In CPA,DAX-1 protein had positive correlation with 24h urinary free cortisol (r=0.709,p=0.022). 4) SF-1 protein in CPA had positive correlation with tumor size(r=0.677,p =0.032).Conclusions:1.In accord with with the first part,SF-1 and DAX-1 protein was different in APA,CPA,NFA and aldosterone-producing carcinoma,which indicated that they might have tight correlation with adrenal tumorigenesis.2.The normal protein expression of SF-1,lower expression of DAX-1,higher SF-1/DAX-1 ratio in AⅡ-U-APA tissues and negative correlation of the ratio with tumor size is also suggested that low DAX-1 might affect the development of AⅡ-U-APA.3.SF-1 and DAX-1 protein also tended to be overexpressed in NFA and aldosterone-secreting adrenocortical carcinoma,which indicated that they could participate the tumorigenesis of aldosterone-secreting adrenocortical carcinoma,and we should also monitor the NFA patients.4.The lower expression of SF-1 in CPA had positive correlation with tumor size,which indicated than SF-1 might have tight correlation with tumorigenesis.5.Some outcomes had not been reported as yet.