The Effect Of Cell Proliferation And Aldosterone Secretion On Adrenal Cortical Adenocarcinoma H295R Cells By Targeted Silencing CTNNB1

Objective: CTNNB1 gene mutation was closely associated with the clinical phenotype of patients with primary aldosteronism(PA).The tumor size of aldosterone adenomas(APA)containing the CTNNB1 gene mutant was larger than that containing the KCNJ5 gene mutant,and the expression of CYP11B2 was also higher,suggesting that the Wnt/β-catenin signaling pathway may play an important role in the formation of APA and the secretion of aldosterone.Therefore,this study intended to explore the effect of CTNNB1 gene on the cell proliferation and aldosterone secretion of adrenal cortical adenocarcinoma H295 R cells based on cellular experiments,and provide a basis for exploring the possible pathogenesis of APA.Methods: RNAi technology was used to target silencing the CTNNB1 gene using pGLMV-SC5 RNAi lentivirus as vector.The experiment was divided into four groups: No-treated group(Control),treated with silencing the CTNNB1 gene(β-catenin)group(si-β-catenin),angiotensin Ⅱ-stimulated control group(Control+Ang Ⅱ),angiotensin Ⅱ-stimulated si-β-catenin group(si-β-catenin+Ang Ⅱ).The growth and fluorescence expression of cells in each group were observed by fluorescence microscope and inverted microscope;The protein and mRNA expression levels of CYP11B2,CYP11B1,AXIN2,LEF1 and Cyclin D1 in H295 R cells were detected by Western blot and qRT-PCR;The expression levels of aldosterone in the cell supernatant of each group were detected by ELISA;The cell proliferation abilities of each group were detected by CCK-8 and cell colony formation assay.The experimental data were collected and collated,and statistical analysis was performed using SPSS13.0.Results: Targeting silencing the CTNNB1 gene successfully down-regulated the expression of β-catenin in adrenal cortical adenocarcinoma H295 R cells.The mRNA and protein expression levels of β-catenin in the si-β-catenin group were decreased significantly compared with the Control group(P<0.001).Compared with the Control group,the mRNA expression levels of AXIN2 and LEF1 in the si-β-catenin group were significantly decreased(P<0.01)and the western blot results showed that the protein levels of AXIN2 and LEF1 in the si-β-catenin group were also decreased significantly.The expressions of AXIN2 and LEF1 in the si-β-catenin+Ang Ⅱ group were further decreased compared with the Control+Ang Ⅱ group,suggesting that targeting silencing the CTNNB1 gene reduced the activity of the Wnt/β-catenin signal pathway.In addition,compared with the Control group,the mRNA expression level of CYP11B2 in the si-β-catenin group was decreased(P<0.001).Under angiotensin Ⅱ stimulation,the mRNA expression level of CYP11B2 in the Control+Ang Ⅱ group was significantly higher than that in the si-β-catenin+Ang Ⅱ group(P<0.001).However,there was no significant difference in the mRNA expression level of CYP11B1 between the Control group and si-β-catenin group(P>0.05).After stimulation with angiotensin Ⅱ,the CYP11B1 mRNA expression levels in the Control+Ang Ⅱ group and the si-β-catenin+Ang Ⅱ group were both increased,but there was no significant difference between the two groups(P>0.05).Western blot analysis showed that silencing CTNNB1 gene significantly decreased the expression of CYP11B2,but had no significant effect on the expression of CYP11B1.The expression level of aldosterone in the supernatant of each group in H295 R cells showed,Control group: 51.477±1.977 ng/ml,si-β-catenin group: 48.830±1.557 ng/ml,Control+Ang Ⅱ group: 80.938±3.092ng/ml,si-β-catenin+Ang Ⅱ group: 56.161±2.725 ng/ml.The expression of aldosterone in the si-β-catenin group decreased 12.91% compared with the Control group(P<0.05);Under angiotensin Ⅱ stimulation,the expression of aldosterone in the si-β-catenin+Ang Ⅱ group decreased 30.61% compared with the Control+Ang Ⅱ group(P<0.001).Moreover,Cyclin D1 is a key protein that regulates cell cycle and affects cell proliferation.The mRNA expression level of Cyclin D1 in the si-β-catenin group was significantly lower than that in the Control group(P<0.01).After angiotensin Ⅱ stimulation,the mRNA expression level of Cyclin D1 in si-β-catenin+Ang Ⅱ group was significantly lower than that in Control+Ang Ⅱ group(P<0.01),but there was no significant difference between the Control+Ang Ⅱ group and the Control group(P>0.05).Western blot analysis showed that silencing CTNNB1 gene significantly reduced the protein expression level of Cyclin D1.The CCK-8 assary showed that the ability of cell proliferation in the si-β-catenin group was significantly lower than that in the Control group(P<0.01).And the cell colony formation experiment showed that the number of cell clones in the si-β-catenin group was significantly less than that in the Control group(P<0.01).Conclusions: β-catenin is the key molecule of Wnt pathway.Targeting silencing CTNNB1 gene reduces the synthesis of β-catenin and decreases the activity of Wnt/β-catenin signaling pathway.Targeting silencing CTNNB1 gene can significantly reduce the expression of CYP11B2 and aldosterone secretion as well as the reactivity of H295 R cells to angiotensin Ⅱ,but has no significant effect on the expression of CYP11B1.Targeting silencing CTNNB1 gene reduces β-catenin-dependent LEF/TCF transcriptional activity and decreases the expression of downstream Cyclin D1,thereby preventing cells from G1 phase to S phase,ultimately inhibiting the proliferation of H295 R cells.