Effect Of KCNJ5 Gene Mutation On Aldosterone Secretion Of Human Adrenal Cortical Carcinoma H295R Cells

Objective:To observe the effects of mutant KCNJ5 G151R and delI157 genes on aldosterone secretion of human adrenal cortical carcinoma cell line H295R by using Lipofectamine^TM3000 transfection technique.Methods:Wild-type KCNJ5,mutant KCNJ5 g151r,KCNJ5delI157 and empty vector plasmid were constructed.Transfected by Lipofectamine^TM3000,the H295R cells were divided into 4 groups,which were wild type KCNJ5 group,mutant KCNJ5 G151R group,mutant KCNJ5 delI157 group and control group.After transfection of plasmid liposome complex into H295R cells,the medium was replaced by those with and without 100n M angiotensin II,as well as those with and without10uM verapamil.Then these cells would be placed in 37?and 5%CO₂cell incubator for 36 hours.The changes of cell membrane potential in each group were detected by DiSBAC2 fluorescent dye.The expression of KCNJ5 protein in each group of cells were detected by protein blot?Western blot?method.The concentration of aldosterone in cell culture supernate of each group were detected by radioimmunoassay.Real-time fluorescence quantitative PCR?Quantitative real-time PCR?would be used to detect the 11-?hydroxylase?CYP11B1?and Aldosterone synthase?CYP11B2?gene expression level.Results:?1?Compared with the control group,the fluorescence intensity of transfection KCNJ5G151R and KCNJ5 del I157 were significantly increased?P<0.05?.?2?The expression of KCNJ5 protein in empty vector plasmid transfected cells,wild type KCNJ5 transfected cells,mutant KCNJ5 G151R transfected cells,and mutant KCNJ5 delI157 transfected cells was1.08±0.04,1.64±0.07,1.82±0.04,1.87±0.05.The expression of KCNJ5protein in the wild-type KCNJ5 group,mutant KCNJ5 G151R group and mutant KCNJ5 delI157 group was significantly higher than that in the control group by t-test.The difference was statistically significant?P<0.05?.There was no significant difference between the mutant KCNJ5G151R group and the KCNJ5 delI157 group?P>0.05?.?3?Detected by radioimmunoassay,the aldosterone concentration of cell culture supernate in the transfectedKCNJ5 G151R mutant group was 2.86 times than those in the control group?1.720±0.045vs0.600±0.039ng/ml,P<0.05?,transfected KCNJ5 delI157 mutant group was 2.26 times than control group?1.356±0.036vs0.600±0.039ng/ml,P<0.05?,while there was no significantly difference between transfected wild type KCNJ5 group and control group?0.636±0.053vs0.600±0.039ng/ml,P>0.05?.?4?The expression of CYP11B1 mRNA of the control group,the wild type KCNJ5 group,the mutant KCNJ5 G151R group and the mutant KCNJ5delI157 group was 1±0.439,2.244±0.774,4.404±0.820,3.089±0.691.The expression of CYP11B1 mRNA of the transfected KCNJ5 G151R group and the mutant KCNJ5 delI157 group was significantly higher than that in control group by t-test?P<0.05?.There was no significant difference between the transfection wild-type KCNJ5 group and the control group?P>0.05?.?5?The expression of CYP11B2 mRNA in control group,wild type KCNJ5 group,mutant KCNJ5 G151R Group and mutant KCNJ5 delI157 Group was 1±0.106,1.302±0.275,2.644±0.063,1.736±0.269.The expression of CYP11B2 mRNA in the transfected KCNJ5 G151R group and the KCNJ5 delI157 group was significantly higher than that in the control group?P<0.05?.There was no significant difference between the wild-type KCNJ5 transfection group and the control group?P>0.05?.?6?aldosterone secreted by transfected mutant KCNJ5 G151R cells increased 1.39 times after angiotensin?'s stimulation?2.381±0.051vs1.704±0.045ng/ml,P<0.05?,which was 1.67times increased in transfected mutant KCNJ5 del1I57 cells2.263±0.042vs 1.349±0.046ng/ml,P<0.05).The expression of CYP11B1 mRNA and CYP11B2 mRNA increased in transfected mutant KCNJ5 G151R cells?1.000±0.036 vs 1.466±0.161,P<0.05?,?1.000±0.066 vs1.806±0.166,P<0.05?.The expression of CYP11B1 mRNA and CYP11B2 mRNA increased in transfected mutant KCNJ5 del1I57 cells?0.796±0.104 vs 1.576±0.064,P<0.05?,?0.867±0.073 vs 2.116±0.052,P<0.05?.?7?verapamil made the aldosterone secretion of mutant KCNJ5G151R and KCNJ5 delI157 cells respectively decrease 37.9%?1.720±0.045vs1.068±0.034ng/ml,P<0.05?and26.8%?1.356±0.036ng/mlvs0.992±0.046,P<0.05?.The expression of CYP11B1 mRNA and CYP11B2 mRNA decreased in transfected mutant KCNJ5G151Rcells?4.044±1.020vs2.455±0.574,P<0.05?,?2.664±0.527 vs 1.266±0.401,P<0.05?.The expression of CYP11B1 mRNA and CYP11B2 mRNA increased in transfected mutant KCNJ5 del1I57 cells?8.053±1.038 vs 3.089±0.691,P<0.05?,?2.623±0.313 vs 1.736±0.469,P<0.05?.Conclusion:?1?KCNJ5 G151R and KCNJ5 del I157 could make the H295R cell membrane potential depolarize,which could increase the expression of CYP11B2 gene and the secretion of aldosterone.?2?The expressions of CYP11B2 mRNA,as well as the aldosterone secretion of transfected mutant KCNJ5 G151R and KCNJ5 delI157 in H295R cells would increase further the Angiotensin?stimulation.?3?The aldosterone secretion of transfected mutant KCNJ5 G151R and KCNJ5 delI157 in H925R cells,as well as the expression of CYP11B2 mRNA in H295R cells would be inhibited after the verapamil's intervening.