The Role And Mechanism Of KSR2 In Abnormal Androgen Secretion And Proliferation In Virilizing Adrenal Cortical Adenomas

A case report of virilizing adrenal cortical adenoma and analysis of gene differential expressionObjectiveVirilizing adrenal cortical adenoma is a rare clinical disease,and its pathogenesis is still unclear.We conducted transcriptome sequencing on the tumor and adjacent tissues of virilizing adrenal cortical adenoma to explore the molecular mechanism of abnormal secretion of androgens.MethodsIn 2015,a female patient with virilizing adrenocortical adenoma in our hospital was closely followed up,and the hormone levels were recorded completely,and transcriptome sequencing was performed on the tumor and adjacent tissues after surgery.The sequencing results were analyzed by bioinformatics to analyze the differentially expressed genes and the biological functions of the differential genes.ResultsA middle-aged woman who was admitted to the hospital due to infertility for 14 years.Laboratory tests in our hospital found serum testosterone(T),dehydroepiandrosterone sulfate(DHEA-S)and urine 17-Ketocorticoids increased significantly.The imaging examination revealed that the left adrenal gland was occupying space,and there was no obvious abnormality in the ovary and pituitary.Postoperative pathology showed:adrenal cortex adenoma.Three years later,she developed pain in the right lower abdomen,and a CT scan revealed a right adrenal nodule.Laboratory tests showed elevated serum testosterone(T)and biological activity T.Postoperative pathology revealed adrenal cortical adenoma with nodular hyperplasia.A total of 239 genes were up-regulated in the first sequencing,and 862 genes were down-regulated.Among them,the expression of genes related to stimulation,CYP1A2,AKR1C1 and AKR1C2,decreased,while the expression of CTAG2,TEX15,CYP1B1 and GNRHR genes increased;the second sequencing results showed that 134 genes were up-regulated and 809 genes were down-regulated.In addition,whole transcriptome sequencing of tissue samples after recurrence showed that 286 lncRNA expressions were up-regulated,251 lncRNA expressions were down-regulated;39 cirRNA expressions were up-regulated,60 cirRNA expressions were down-regulated;112 miRNA expressions were up-regulated,and 153 miRNA expressions were down-regulated.ConclusionsVirilizing adrenal cortical adenoma is a disease that has a serious impact on the development of female secondary sexual characteristics and fertility.The sequencing results show that there is no differential expression of genes involved in androgen synthesis,indicating that other genes or post-transcriptional regulation may be involved in the regulation process of androgen synthesis.Part ⅡThe mechanism of KSR2 gene regulating androgen secretion and proliferation in virilizing adrenal cortical adenomasObjectiveThe sequencing results of virilizing adrenocortical adenoma patient showed that the expression of KSR2 gene decreased,suggesting that KSR2 may be related to abnormal secretion and proliferation of androgens in the disease.This part of the study uses NCL-H295R cells as a model to explore the molecular mechanism of KSR2 regulating androgen secretion and proliferation,with a view to finding new molecular targets for the diagnosis and treatment of virilizing adrenal cortical adenomas.MethodsAnalyze the expression of KSR2 protein in non-functional adrenal cortical adenoma,virilizing adrenocortical adenoma,adrenal aldosteronoma,and adrenocortical carcinoma by immunohistochemistry,knocked down KSR2 lentivirus to infect NCL-H295R cells,and detected DHEA and DHEA by ELISA-S level,and then infected NCL-H295R cells with overexpressing KSR2 lentivirus,ELISA to detect the levels of DHEA and DHEA-S;then after knocking down KSR2 gene,RT-PCR detected adrenal androgen synthesis related genes(SATR,CYP17A1,HSD3B2,SULT2A1,SF-1,DAX-1,AKR1C3,CYP11A1)expression,Western blot detection of adrenal androgen synthesis related protein(CYP17A1,HSD3B2)expression level.Download the TCGA database adrenal cortical cancer clinical data,analyzed the correlation between androgen secretion and adrenal tumors,then used Celigo and MTT to detect cell proliferation,flow cytometry to detect cell cycle and apoptosis,and plate cloning to detect cell cloning.ResultsImmunohistochemical results showed that the expression of KSR2 in virilizing adrenal cortical adenomas was lower than that of other adrenocortical tumors.After knocking down the KSR2 gene expression in NCL-H295R cells,the ELISA results showed that the secretion of DHEA and DHEA-S was increased.After the overexpressing of KSR2 gene,the secretion of DHEA and DHEA-S was reduced.RT-PCR detection showed that inhibition of KSR2 increased the expression of adrenal androgen synthesis related genes CYP17A1 and HSD3B2.Western blot results also confirmed that the protein levels of CYP17A1 and HSD3B2 increased after KSR2 was inhibited;conversely,the protein levels of CYP17A1 and HSD3B2 decreased after KSR2 was overexpressed.Data analysis of adrenocortical carcinoma in the TCGA database found that androgens were negatively correlated with the prognosis of adrenocortical carcinoma.Celigo test results showed that inhibition of KSR2 cell proliferation was weakened,and MTT analysis confirmed that KSR2 could promote cell proliferation;after inhibiting KSR2,the number of apoptotic cells was significantly increased,the cloning ability was weakened,the cell cycle was arrested,and the expression of Cyclin D1 and CDK4 related to the cell cycle decreased.And the proliferation ability of overexpression is significantly enhanced.ConclusionsKSR2 protein is low expressed in virilizing adrenal cortical adenomas,while KSR2 is negatively correlated with adrenal androgen synthesis in NCL-H295R cells,and overexpression of KSR2 can inhibit the expression of adrenal androgen synthesis-related genes CYP17A1 and HSD3B2.At the same time,KSR2 can inhibit the apoptosis of NCL-H295R cells and promote their proliferation and cloning ability.Part ⅢKSR2 gene regulates androgen secretion and proliferation of virilizing adrenal cortical adenomas through the ERK signaling pathwayObjectiveTo explore the molecular mechanism of KSR2 gene regulating androgen secretion and proliferation of virilizing adrenal cortical adenomas through the ERK signaling pathway.MethodsThe expression of ERK1/2,CYP17A1,HSD3B2 protein in non-functional adrenal cortical adenoma,virilizing adrenal cortical adenoma,adrenal aldosteronoma,and adrenocortical carcinoma were analyzed by immunohistochemistry.The STRING database analyzed the protein interaction of KSR2,and then knocked down and overexpressed the KSR2 gene of NCL-H295R cells respectively.Western blot detected the expression changes of ERK1/2 and phosphorylated ERK1/2(p-ERK1/2),and immunofluorescence analysised the localization of p-ERK1/2 in NCL-H295R cells.NCL-H295R cells were treated with ERK inhibitor U0126,the changes of DHEA and DHEA-S concentrations were detected by ELISA,and the expression of ERK1/2,p-ERK1/2,CYP17A1 and HSD3B2 were detected by Western blot.At the same time,the protein kinase A signal pathway activator 8Br-cAMP was used to treat NCL-H295R cells,the changes of DHEA and DHEA-S concentration were detected by ELISA,and the expression of ERK1/2,p-ERK1/2,KSR2,CYP17A1,and HSD3B2 were detected by Western blot.U0126 treated NCL-H295R cells overexpressing KSR2,MTT detects cell proliferation,flow cytometry detected cell cycle and apoptosis,and plate cloning test detected cell cloning ability.ResultsThe results of immunohistochemistry showed that the expression of ERK and CYP17A1 in virilizing adrenocortical adenoma tumor tissue was significantly increased,and there was no significant difference in the expression of HSD3B2.STRING database analysis showed that KSR2 interacted closely with the MAPK signaling pathway.After knocking down the KSR2 gene,the expression of ERK protein did not change significantly,and the expression of p-ERK 1/2 decreased;over-expression of the KSR2 gene,the expression of p-ERK1/2 increased,immunofluorescence showed that the nuclear localization of p-ERK1/2 decreased after knocking down the K SR2 gene.After U0126 treatment,ELISA results showed that DHEA and DHEA-S secretion increased,Western blot results showed that the expression of p-ERK1/2 decreased,and the expression of CYP17A1 and HSD3B2 increased;while U0126 could partially reversed the p-ERK1/2 caused by overexpression of KSR2 The expression increased,and the expression of CYP17A1 and HSD3B2 decreased.8Br-cAMP could promote the secretion of DHEA and DHEA-S,inhibit ERK1/2 phosphorylation,and 8Br-cAMP could inhibit the expression of KSR2 protein.At the same time,U0126 could partially inhibit the increased proliferation of NCL-H295R cells caused by overexpression of KSR2 and the decrease of apoptosis.ConclusionsKSR2 gene can promote the phosphorylation of ERK1/2 of NCL-H295R cells and its transport to the nucleus;inhibition of the ERK pathway can promote the expression of CYP17A1,HSD3B2 and the secretion of HEA and DHEA-S;KSR2 can promote the proliferation of NCL-H295R cells through the ERK signaling pathway.