Expression Of CYP11B₁、CYP11B₂and ACTH-R In Human Adrenal Tumor Tissues

OBJECTIVES:The purpose of this investigation was to analyze the level of expression of CYP11B1、CYP11B2and ACTH-R mRNAs in normal adrenal cortex, aldosterone producing adenomas, cortisol producing adenomas, and pheochromocytoma to explore the relationship between the expression of these three genes and the pathogenesis of the adrenal tumors mentioned above.METHODS:1. Total RNA of normal adrenal cortex (n=6), aldosterone producing adenomas (n=18), cortisol producing adenomas (n=11), and pheochromocytoma (n=2) were extracted using Trizol agent.2. PCR method was employed after the reverse transcription to amplify the cDNA of CYP11B1, CYP11B2, ACTH-R and endogenous reference GAPDH. The products went through electrophoresis and the grey intensities of the bands were calculated using the software. The ratios of the grey intensities, as CYP11B1/GAPDH、CYP11B2/GAPDH and ACTH-R/GAPDH were analyzed to indicate the relative amount of production of the certain genes.3. Comparisons were made among the gene expression of CYP11B1、CYP11B2and ACTH-R in different adrenal tissues respectively. The analysis of the relations between the CYP11B1、CYP11B2and ACTH-R mRNA levels and the clinical indices as the tumor volume and hormonal level was also carried out. Study was also engaged in the exploration of the relationship among the expression of these three genes in different adrenal tumor tissues.RESULTS:1. All three genes (CYP11B1、CYP11B2and ACTH-R) studied ware expressed at various levels in different adrenal tissues as normal adrenal cortex, aldosterone producing adenomas and cortisol producing adenomas. None of the genes was found to be expressed in the tissues of pheochromocytoma.2. The study of CYP11B1、CYP11B2and ACTH-R genes on aldosterone producing adenomas1) The expression of CYP11B1、CYP11B2and ACTH-R genes in aldosterone producing adenomasCYP11B1was minimally expressed in aldosterone producing adenomas (0.65±0.34) when compared with those in normal adrenal cortical tissues (0.72±0.13,P<0.01) and cortisol producing adenomas (0.99±0.50, P<0.01).CYP11B2was maximally expressed in aldosterone producing adenomas (1.55±0.43) when compared with those in normal adrenal cortical tissues (1.25±0.75, P<0.01) and cortisol producing adenomas(1.24±0.52,P<0.01).The expression of ACTH-R in aldosterone producing adenomas (0.62±0.27) was lower than those in the normal adrenal cortical tissues (0.98±0.36, P<0.01) as well as the cortisol producing adenomas (0.87±0.28,P<0.01).2) Correlation of CYP11B1、CYP11B2and ACTH-R with clinical indications of APA patients:In the aldosterone producing adenomas, CYP11B1mRNA levels were not-correlated with potassium or sodium levels in the blood. There was also no significant correlation between the CYP11B1expression with rennin activities, angiotensin and aldosterone concentrations in serum and24h UFCs.In the aldosterone producing adenomas, CYP11B2mRNA levels were negatively correlated with the potassium levels (r=-0.718, p=0.04) and positively correlated with the aldosterone level at the standing position (r=0.708, p=0.045) in hypokelamic patients(n=8). No significant relations were found between the expressions of CYP11B2genes with any of the factors as sodium, rennin, angiotensin, recumbent aldosterone and24h UFC levels in hypokelamic patients. For the patients with normal potassium level (n=10), no correlations were found between the CYP11B2expression and clinical indices.For the rennin reactive APA patients (n=3), ACTH-R mRNA levels(0.54±0.14) were markedly lower than the non-reactive ones (n=15)(0.64±0.25P<0.01).In ACTH-R positive APA patients (n=11), ACTH-R mRNA is positively correlated with the aldosterone level at the standing position (r=0.845, P<0.01), but were not correlated with any of the factors as sodium, potassium levels, rennin activities, angiotensin, recumbent aldosterone concentrations and24h UFC levels.3) Correlation studies of the gene expressions between CYP11B1、CYP11B2and ACTH-R in APAs. The expressions of CYP11B2gene was positively correlated with the ACTH-R mRNA levels of all APA patients.(r=0.662, p<0.01) The expressions of CYP11B2gene was positively correlated with the ACTH-R mRNA levels of ACTH-R positive APA patients.(r=0.765, P<0.01)3. The study of CYP11B、CYP11B2and ACTH-R in cortisol producing adenomas1) The expression of CYP11B1、CYP11B2and ACTH-R genes in cortisol producing adenomasCYP11B1was maximally expressed in cortisol producing adenomas(0.99±0.50) when compared with those in normal adrenal cortical tissues (0.72±0.13, P<0.01) and aldosterone producing adenomas (0.65±0.34, P<0.01). CYP11B2was minimally expressed in cortisol producing adenomas(1.24±0.52) when compared with those in normal adrenal cortical tissues (1.25±0.75, P=0.01) and aldosterone producing adenomas (1.55±0.43, P<0.01). The expression of ACTH-R in cortisol producing adenomas (0.87±0.28) was lower than those in the normal adrenal cortical tissues (0.98±0.36, P<0.01) but higher than the aldosterone producing adenomas (0.62±0.27, P<0.01)2) Correlation of CYP11B1、CYP11B2and ACTH-R with the following clinical indications in cortisol producing adenomas. In the cortisol producing adenomas, CYP11B1mRNA levels were positively correlated with24h UFCs (r=0.815, p=0.002) but not the total cortisol concentrations (r=0.537, p=0.068) or ACTH levels in blood.In the cortisol producing adenomas, CYP11B2mRNA levels were not statistically relevant to the24h UFC levels, total cortisol concentrations or ACTH levels in blood.ACTH-R mRNA levels in cortisol producing adenomas were negatively correlated with24h UFC levels (r=-0.629, p=0.038). There was no meaningful relation found between the gene expressions with the total cortisol concentrations.3) Gene expressions of CYP11B1、CYP11B2and ACTH-R were not correlated with each other in cortisol producing adenomas.CONCLUSIONS:1. CYP11Biwas markedly expressed in the cortisol producing adenomas and the level of which was positively correlated with24h UFC of patients. This fact found in the experiment indicates CYP11Bi might play an important role in the hormone production of cortisol producing adenomas.2. CYP11B2was markedly expressed in the aldosterone producing adenomas and the level of which was negatively correlated with potassium level and positively correlated with the aldosterone level of hypokelamic patients. This fact found in the experiment indicates CYP11B2might play a significant role in the hormone production of aldosterone producing adenomas.3. The expression of ACTH-R declined in cortisol producing adenomas which suggested that cortisol production in cortisol producing adenoma is suppressed. ACTH-R expression was higher in the rennin responsive APA patients than the non-responsive ones to indicate the possible effect of ACTH to the responsiveness of APA to RAS system. The expression of ACTH-R and CYP11B2were positively correlated in APA patients and the ACTH-R mRNA levels were relevant to the aldosterone concentration of the APA patients. This fact suggested that the hormone production of APA patients may be regulated by ACTH through its influence on CYP11B2production.4. CYP11B1、CYP11B2and ACTH-R were all not found expressed in pheochromocytoma which indicated they were not relevant to the pathogenesis of pheochromocytoma.5. According to the literature review of MEDLINE, some of the results in this study are reported for the first time.