The Role And Mechanism Of Estrogen-SOX2 Pathway In Cushing’s Syndrome During Pregnancy

BackgroundCushing’s syndrome(CS)during pregnancy is a rara acute and critical disease in endocrinology department which can cause serious harm to the mother and fetus,and even endanger life.At present,the pathogenesis of CS during pregnancy is not clear,and there is a lack of effective intervention in treatment,which is a particularly important clinical problem to be solved urgently.In pregnancy with CS,cortisol-producing adrenocortical adenoma(CPA)is the main cause.Case reports shows that the majority patients have no typical clinical manifestations of CS before pregnancy.CS develops rapidly and progresses during pregnancy,and spontaneously alleviates with the end of pregnancy.Most studies indicated that an aberrant expression of luteinizing hormone(LH)/human chorionic gonadotropin(hCG)receptor(LHCGR)in CPA played a critical role in the pathogenesis and exacerbation of CPA during pregnancy.However,the pathogenesis does not fully explain the clinical problems.Estrogen is an important pregnancy-related sex hormone,sustained exposure to high levels of estrogen is a risk factor for many cancers.It has been reported that estrogen receptor α(ERα)is highly expressed in adrenal cortical adenocarcinoma cells,and the combination of estradiol(E2)with it can promote the proliferation of adrenal cortical adenocarcinoma cells.These findings suggest that the activation of estrogen and its receptor ERa pathway may play a key role in CS during pregnancy,but the mechanism remains to be further explained.Specific environmental factors can induce epigenetic modification and participate in gene space-time regulation and cell differentiation.Epigenetic alterations have also been reported in adrenal Cushing.Considering the great changes of environment and hormones during pregnancy,CS during pregnancy may also have epigenetic modification abnormalities.In order to explore the role and mechanism of estrogen and its receptor ERa in CS during pregnancy,we intend to culture adrenocortical adenocarcinoma cells in vitro to investigate the effect of estrogen on their proliferation.The expression of ERa protein was detected in pregnant and non-pregnant CPA tissue samples.Advanced methylation chip technology was used to map the whole genome of pregnant and non-pregnant CPA patients,and further bioassay was performed to screen out target genes and conduct preliminary functional studies.Part 1 The role of estrogen in the occurrence and development of pregnancy with Cushing’s syndromeObjectiveTo investigate the effect of estrogen on proliferation of human adrenocortical adenocarcinoma cells,and to detect the expression of ERa protein in pregnant and nonpregnant CPA tissue samples.Methods1.Clinical data of pregnant patients with CS(pregnancy group)and non-pregnant patients with CS(non-pregnancy group)were collected by electronic medical record system,and the differences of related indicators between the two groups were compared.2.Immunohistochemical staining(IHC)was performed to observe the expression level of ERa in pregnant and non-pregnant CPA tissues.3.Human adrenocortical adenocarcinoma cell H295R was cultivated in vitro and stimulated with 17β-E2 at different concentrations and for different times.then cell proliferation was measured by CCK8.Results1.Compared with clinical characteristics of CPA patients without pregnancy,the pregnant patients with CPA had higher serum hCG,E2 and cortisol concentration(P<0.05),No significant difference in tumor size was found between the non-pregnant and pregnant group(P=0.83).Furthermore,compared with the non-pregnant patients,the duration is shorter,and the growth rate of the tumor is faster in pregnant patients with CPA(P<0.05).2.IHC showed that ERa expression in CPA tumor tissue was significantly higher than that in adjacent tissue(P<0.005),and the pregnant group had higher levels of ERα than nonpregnant adenomas group(P<0.005).3.Compared with the control group,the proliferation level of H295R was significantly increased after stimulated with 17β-E2 at different concentrations and at different times(P<0.05).ConclusionERa pathway was activated in the tumor tissue of patient of CS during pregnancy.Moreover,vitro studies have also shown that estrogen can significantly promote the proliferation of human adrenocortical cancer cells.These suggest that the activation of estrogen and its receptor ERa pathway may play a key role in CS during pregnancy.Part 2 The role and mechanism of estrogen-SOX2 pathway in Cushing’s syndrome during PregnancyObjectiveTo investigate the role and mechanism of estrogen in CS during pregnancy.Methods1.Genome-wide methylation maps of pregnant and non-pregnant CPA patients were mapped by methylation chip technology,and target genes were screened by bioassay.2.IHC was performed to observe the expression level of SOX2 in pregnant and nonpregnant CPA tissues.3.Constructed the target gene overexpression plasmid,and then transfected into H295R cells.CCK-8 assay was used to determine the effect of target genes.on cell proliferation.4.Stimulated H295R cells by 17β-E2,and the protein and mRNA expressions of target genes were detected by Western blot and RT-qPCR.Results1.Methylation analysis showed that there were significant differences in the genomewide methylation profiles between pregnant and non-pregnant CPA patients.Target gene SOX2 was screened by GO functional analysis and KEGG pathway enrichment.2.IHC showed that SOX2 expression in CPA tumor tissue was significantly higher than that in adjacent tissue(P<0.05),and the pregnant group had higher levels of SOX2 than nonpregnant adenomas group(P<0.05).3.SOX2 overexpression plasmid was constructed and successfully transfected into H295R cells.Compared with the control group,the proliferation level of SOX2 overexpression group was significantly increased(P<0.001).4.Compared with the control group,expression of SOX2 protein and gene were significantly increased after stimulated with 17β-E2(P<0.0001).ConclusionSignificant differences in the whole genome methylation map between pregnant and non-pregnant CPA patients,suggested that abnormal DNA methylation modification may be involved in the occurrence and development of CS during pregnancy.Estrogen may be involved in regulating the proliferation of adrenocortical cancer cells through the SOX2 pathway.