Emotional Stress Accelerated Ovarian Tumor Transfer Through GR/NUPR1 Regulation

Background:Emotion inducing diseases is an important part of etiology and pathogenesis theory in Traditional Chinese Medicine.Emotional stress is a series of mental and emotional responses of the body to objective things and phenomena from the outside world,including mental reactions and functional activities of the viscera.We have established virus-susceptibility model and tumor-susceptibility model by emotional stress early on,it was found that emotional stress can up-regulate the concentration of corticosterone in nude mice,and then facilitate the incidence of influenza and tumor metastasis.Tumor belongs to the ?Ji? in Traditional Chinese Medicine,and its susceptibility is closely related to emotions.Huang di's Canon of Medicine also follows the sentence,?When anger and sorrow cause internal injuries,the ?Qi?inverses,forming ?Ji??.According to the traditional Chinese medicine theory,the main pathogenies of gynecological ovarian tumor is tangible evil of ?Qi? stagnation,congealing cold and etc.on the lower abdomen,transforming into ?Ji? with time passing.The mortality of ovarian tumor is the highest among gynecological tumors,and the incidence is increasing year by year,which seriously threatens women's health.Epidemiological investigation reported that work stress and negative emotion can facilitate the metastasis of ovarian tumor.The modern medical model and the traditional Chinese medicine theory have made a consensus that the emotional factors play a significant role in tumor induction.Emotional stress stimulates the hypothalamic-pituitary-adrenal(HPA)axis to release multiple hormones constituting the psycho-neuroendocrine regulatory system,and the released glucocorticoid activates the glucocorticoid receptor(GR).Preliminary studies have reported that the GR expression in ovarian tumor samples negatively correlate with Progression-Free-Survival of patients with ovarian tumor.However,how GR is involved in ovarian invasion and metastasis has not been well reported.Therefore,the study on the regulation of psychology-neuroendocrinology on ovarian tumor metastasis and its molecular mechanism are of great importance.It is helpful for elucidating the pathological mechanism of ovarian tumor metastasis and is expected to provide basis for exploring the treatment strategy of ovarian tumor metastasis and recurrence.Objective:The mechanism of emotional stress facilitating ovarian tumor metastasis was studied using an in vivo model of ovarian tumor metastasis loaded with emotional stress,combining with pathological characteristics analysis of clinical samples and data obtained from in vitro cell model.In order to explore new therapeutic targets of anti-ovarian tumor metastasis,necessary theoretical and experimental foundation of new strategies to prevent and treat ovarian tumor recurrence and metastasis will be provided.Method:In vivo experiments: Restraint stress and Corticosterone loading stress were used to simulate emotional stress induced tumor cell metastasis in nude mice.Firstly,the in vivo imaging system was used to observe the colonization of metastatic tumors and metastatic foci of internal organs in nude mice,and pathological morphology of liver metastases was detected by HE;then,serum Corticosterone and CA125 were detected by ELISA;the interest protein expression were detected by IHC.Clinical data analysis: Firstly,MRI technique was used to observe the morphology of ovarian tumor,normal ovarian and ovarian cysts,and then,the expressions of GR and NUPR1 in 3 types of ovarian samples were detected by IHC.SPSS software was used for statistical analysis of the correlations between the expression of GR and NUPR1 in tumor tissues of patients and various clinicopathological characteristics;then,Oncomine database was used to analyze the difference of NUPR1 mRNA expression level between ovarian tumors and normal ovarian;cBio Portal database was used to analyze the correlations of the GR(NR3C1),NUPR1 and downstream regulate gene Slug(SNAI2)mRNA expression level in clinical samples;Finally,Kaplan Meier-plotter database was used to analyze the correlations of NR3C1,NUPR1 mRNA level and PFS?In vitro experiments:First,a Corticosterone-induced ovarian tumor migration and invasion cell model was established.The ability of cell invasion and migration was evaluated using Scratch and Transwell methods;then,expression profile microarray were used to analyze the difference in gene expression and IPA software was used to predict the upstream regulator gene as NUPR1;finally,the regulatory relationship between GR/NUPR1 and the regulatory effect of NUPR1 on Slug were detected by IF,WB,gene silencing and other techniques in the cell model.Results:1.Emotional stress activates GR to promote ovarian tumor metastasis.In restraint stress group(Str group),all the mice showed liver and intestinal metastasis,66.7% of the group showed stomach metastasis,and 33.3% of the group showed spleen metastasis.Intestinal metastasis was observed in Corticosterone group,stomach and liver metastasis were observed in 66.7% of the group.In Restraint stress+Corticosterone inhibitor(Str+Int(GR))group,all the mice showed intestinal metastasis,66.7% of the group showed stomach metastasis,however,liver,spleen and kidney metastasis were not observed.Serum levels of Corticosterone and CA125 in Str group were higher than those in the control group(P<0.001).CA125 level in Str+Int(GR)group was lower than that in Str group(P<0.001).Clinical IHC data display normal ovarian tissue present GR low expression(the score?6);GR is highly expressed in 64% of the ovarian tumor tissue;GR is highly expressed in 37% of the Endometriosis ovarian cyst.One-way ANOVA analysis results show that the expression of GR in the ovarian tumor was significantly different from that in non-ovarian tumor tissue(P<0.05).Kaplan-Meier analysis results show that the expression of GR in tumor tissue is exclusivity associated with FIGO stage(P<0.05).2.Mechanism of ovarian tumor cell metastasis induced by activated GR.GR activation promoted the transposition of a cytoskeleton protein,actin,from the cell membrane to the cytoplasm,and hence the invasion and metastasis ability of the cell.Gene expression profile microarray analysis differential gene of CORT treatment group and control group,the result showed that epithelial-mesenchymal transition(EMT)transcription factor SNAI2 increased obviously.IPA predicted that the GR activation induced the increasing ability of cell invasion and metastasis might be regulated by the upstream NUPR1 gene.Sequencing results of 185 cases of ovarian tumors and 10 cases of normal ovarian tissues were analyzed through Oncomine database.1.7 times higher expression of NUPR1 in ovarian tumor tissues than that in normal ovarian epithelial tissues,with statisitical significance(P<0.001).The data of clinical samples we collected showed normal ovarian tissue present GR low expression(the score?6);GR is highly expressed in 59% of the ovarian tumor tissue;GR is highly expressed in 33% of the Endometriosis ovarian cyst.One-way ANOVA analysis results show that the expression of NUPR1 in the ovarian tumor was significantly different from that in non-ovarian tumor tissue(P<0.05).Moreover,a significant exclusively positive correlation between the expression of NUPR1 and GR was revealed with Logistic analysis(P<0.001).Kaplan Meier-plotter database analysis results show that there were negative correlation of NR3C1,NUPR1 mRNA level expression and PFS.3.The role of GR/NUPR1 signaling pathway in ovarian tumor metastasis.CORT promoted NUPR1 mRNA level increase in cells(P<0.001),and si RNA-NR3C1 reversed the high expression of CORT-promoted NUPR1/Slug and low expression of the E-cadherin(P<0.001,P<0.001 and P<0.05).CORT decreased the invasion ability of NUPR1 knockdown cells(P<0.001).Compared with control group,Slug protein decreased and E-Cadherin protein expression increased in NUPR1 knockdown cells(P<0.001).The sequence analysis results of 1766 patients with ovarian cancer tumor through cBio Portal database showed that the abnormal amplification and mutation of GR(NR3C1),NUPR1 and Slug(SNAI2)presented a trend of Mutual exclusivity.Conclusion:1.Emotional stress promoted intraperitoneal ovarian tumor metastasis in nude mice by activating GR.GR expression in clinical ovarian tumor tissue is higher than that in non-ovarian tumor tissue,moreover,GR expression in ovarian tumor tissues was significantly exclusivity correlated with FIGO stage in patients.Emotional stress promoted ovarian tumor metastasis may be related to excessive corticosteroid level induced GR activation.2.In vitro experiments confirmed that activation of GR promote invasion and metastasis of ovarian tumor cells by regulating the expression of related proteins through the NUPR1 gene.Moreover,an exclusive positive correlation between the expression of NUPR1 and GR in clinic ovarian tumor tissue was detected.Therefore,metastasis of ovarian tumor may be related to GR regulated NUPR1.There were negative correlation of NR3C1,NUPR1 mRNA level expression and PFS.3.This study investigated the mechanism of emotional stress promoting ovarian tumor metastasis.It was clarified that emotional stress induces GR/NUPR1 signaling pathway can regulate the SNAI2 and promote the occurrence of EMT in ovarian tumor cells.This paper revealed the regulatory effect of emotional stress on ovarian tumor metastasis from the aspect of psychology-neuroendocrinology,and provided necessary theoretical and experimental basis for elucidation of social psychology as an important factor affecting ovarian tumor metastasis.