The Expression Of BCAR3 Gene In Endometriosis And The Effect Of BCAR3 On The Biological Characteristics Of Eutopic Epithelial And Stromal Cells

Objective: Endometriosis(EM)is a common and refractory gynecological disease.Although EMs is an benign disorder,it has the malignant-like characteristics,including local implantation and invasion and distant metastasis.With symptoms of progressive dysmenorrhea,chronic pelvic pain and infertility,it exerts a huge impact on physical as well as mental well-being of female.After years of researches in the field of EM,people have now realized that EM is an estrogen-dependent multifactorial disease.That altered expression of genes related to cell adhesion and invasion result in the functional changes of eutopic cells and etrograde menstruation provide the basic conditions for ectopic implantation.Abnormal autoimmune function and increased estrogen metabolism in ectopic endometrium also play important roles in the occurrence of endometriosis.he breast cancer anti-estrogen resistance 3(BCAR3)gene belongs to the novel Src homology 2(SH2)-containing protein(NSP)family.It is reported that BCAR3 gene promotes cell migration,invasion,and epithelial-mesenchymal transition(EMT).In addition,the BCAR3 protein is responsible for the resistance of estrogen-dependent breast cancer cells to anti-estrogen drugs in vitro,which is an important cause of the failure of endocrine therapy in estrogen-receptor-positive breast cancer patients.Anti-estrogen in ectopic endometrium is a protective factor against the occurrence of EM,and inhibition of the local anti-estrogenic effect promotes the formation of ectopic lesions.Therefore,the potential role of BCAR3 as a candidate gene promoting the malignant behaviors of cells is worth exploring.The results of DIANA-micro T and Targetscan algorithm showed that BCAR3 gene was one of the downstream targets of mi R-126-5p.Mi R-126-5p and mi R-126-3p are derived from the same precursor,pre-mir-126.They are expressed at the same level in various tissues and cells,have similar variation trends,and play similar functions.The expression of mi R-126-5p and mi R-126-3p are low in many cancers,and they can be used to inhibit the migration and invasion of cancer cells.previous study from our group showed that low expression of mi R-126-3p in eutopic endometrial stromal cells(ESCs)of patients with EM leads to increased invasiveness.Whether mi R-126-5p can regulate the invasiveness of ESCs by targeting BCAR3 remains to be verified.The present study investigated the effect of BCAR3 on the behavers of endometril cells and its possible regulatory mechanism to provide a new theoretical basis for the pathogenesis of EM.Methods: Eutopic endometrium(Eu)and ectopic endometrium(Ec)samples were obtained from 32 patients with EM.Normal endometrium(NE)samples from 31 patients without EM.Quantitative real-time polymerase chain reaction(q RT-PCR),Immunohistochemistry and Western blot detected the expression level of BCAR3 protein and m RNA level,while q RT-PCR applied to detect mi R-126-5p in all tissue samples.Then,correlation was analyzed using the Pearson's correlation test.Immunohistochemical method was used to exam the expression level of E-cadherin and vimentin.Primary culture of eutopic epithelial cells(EECs).Lentivirus packaged human BCAR3 c DNA was transfected into EECs to overexpress BCAR3.The morphological change was analyzed by optical microscopy.Western blot was used to examine the expression of E-cadherin,fibronectin and vimentin.Then,EECs were cultured with estrogen alone or combined with estrogen receptor antagonists.The morphology of the cells was also observed by optical microscopy.Western blot was used to examine the expression of E-cadherin,N-cadherin,fibronectin and vimentin.EECs transfected with BCAR3 were dealt with estrogen or estrogen receptor antagonists.The morphological change was analyzed by optical microscopy.E-cadherin,N-cadherin,fibronectin and vimentin protein were detected by western blot.Primary culture of ESCs and normal endometrium cells(NESCs).Western blot and q RT-PCR and were used to detect the expression of mi R-126-5p and BCAR3 m RNA and protein in ESCs and NESCs.Data then analyzed with Pearson's correlation test.Lipofectamine 2000 was used to transfect mi R-126-5p agomir in ESCs and to transfect mi R-126-5p antagomir in NESCs.After measureing transfection efficiency of mi R-126-5p by q RT-PCR,cell proliferation,migration and invasion was examined by CCK8 or Transwell,moreover,BCAR3 protein was detected by western blot.Lentivirus packaged human BCAR3 c DNA,BCAR3 si RNA were separately transfected into ESCs and NESCs.After measureing transfection efficiency by western blot,cell proliferation,migration and invasion was examined by CCK8 or Transwell.HEK293 T cells were cultured in 24-well plates,then co-transfected with mi R-126-5p agomir or negative controls and wild-type-or mutant-BCAR3 3?-UTR reporter vector.luciferase activity was measured by the Dual-Glo Luciferase Assay System.ESCs,transfecting with pre-mi R-126-5p lentivirus,then transfected with BCAR3 c DNA lentivirus or negative control,while NESCs,transfecting with mi R-126-5p si RNA lentivirus to stably silence mi R-126-5p,then transfected with BCAR3 si RNA lentivirus or negative control.Transfection efficiency of lentivirus was measured with the level of BCAR3 protein and mi R-126-5p by western blot and q RT-PCR.CCK-8 cell proliferation assay and Transwell assay were applied to explore the changes of cell proliferation,migration and invasion.Results: 1.qRT-PCR and western blot showed that BCAR3 m RNA and protein were significantly higher in ectopic endometrium compared with eutopic endometrium(P<0.01,P<0.01),and were significantly lower in normal endometrium than eutopic endometrium(P<0.05,P<0.01).The expression of mi R-126-5p was significantly different between Ec and Eu as well as between Eu and NE,P<0.01.Pearson's correlation analysis showed that mi R-126-5p was negatively correlated with BCAR3 m RNA(r=-0.417,P < 0.01)and BCAR3 protein expression(r=-0.669,P < 0.01).We found that BCAR3 staining was in the cellular nuclei and cytoplasm of the stromal cells as well as epithelial cells.BCAR3 expression was significantly higher in endometriotic epithelial cells(Ec ECs)than in EECs and normal endometrial epithelial cells(NEECs)(P < 0.05),which had similar expression levels.In stromal cells,BCAR3 protein expression was highest in Ec SCs,lower in ESCs,and lowest in NESCs(P < 0.01).Immunohistochemical results showed that vimentin was expressed in both epithelial and stromal cells.The staining for vimentin expression was higher in Ec ECs than in EECs and NEECs(P < 0.01).whereas E-cadherin,an epithelial marker,was only expressed in epithelial cells and not in stromal cells.Its expression was significantly lower in Ec ECs than in EECs(P < 0.01).E-cadherin expression levels were similar in EECs and NEECs(P > 0.05).2.The primary culture of EECs was successful.EECs infected with BCAR3-lentivirus showed significantly increased BCAR3 expression(P < 0.05).EECs was identified by vimentin negative and cytokeratin positive.The opposite result,vimentin positive and cytokeratin negative,was ESCs.Compared with the controls,BCAR3 overexpressing cells showed increased fibronectin expression(P < 0.05,Fig.7B)and mesenchymal-like morphology,characterized by disaggregation of spindle-like cells.However,no significant changes in the expression of E-cadherin and vimentin were observed(P > 0.05).Compared with control cells,estrogen-treated EECs showed decreased expression of E-cadherin(P < 0.05)as well as increased expression of vimentin(P < 0.01),and these changes were abolished by estrogen receptor antagonist.In addition,estrogen treatment changed EEC morphology from tightly-arranged round cells to scattered spindle-like cells.EECs treated with estrogen at 24 h after BCAR3-lentivirus infection showed a higher expression of fibronectin(P < 0.05)and an earlier alteration of cell morphology than those in the estrogen-only intervention group and recombinant BCAR3 group.The expression levels of E-cadherin and vimentin were similar between the estrogen intervention plus recombinant BCAR3 group and the estrogen-only intervention group(P > 0.05).3.The primary culture of endometrial stromal cell was successful.The expression of mi R-126-5p was significantly lower in ESCs than in NESCs(P < 0.01),while BCAR3 m RNA and protein were significantly higher in ESCs compared with NESCs(P < 0.01,P < 0.01).The negative relation was found between the expression of mi R-126-5p and BCAR3 m RNA(r=-0.318,P<0.05)and protein(r=-0.756,P<0.01).The results of q RT-PCR and Western blot showed that mi R-126-5p was significantly up-regulated after transfecting mi R-126-5p agomir in ESCs(P < 0.01),and BCAR3 protein was down-regulated(P < 0.01).mi R-126-5p was significantly lower after transfecting mi R-126-5p antagomir in NESCs(P < 0.01),and BCAR3 protein expression was higher(P < 0.05).The result of CCK8 and Transwell assay showed that,comparing to their negative controls,proliferation,migration and invasion of ESCs were decreased after transfecting mi R-126-5p agomir(P<0.05?P<0.01?P<0.01),while proliferation,migration and invasion of NESCs were increased after transfecting mi R-126-5p antagomir(P<0.05?P<0.01?P<0.01).Then ESCs were infected with si BCAR3-lentivirus and NESCs were infected with BCAR3-lentivirus and BCAR3 protein level in each group was comfirmed by Western blot(P < 0.01).As showed by CCK8 and Transwell assay,compared with the control group,cell proliferation,migration and invasion were markedly suppressed in BCAR3 silenced ESCs and significantly promoted in BCAR3-overexpressing NESCs.We then performed luciferase activity assays in HEK293 T cells.The results showed that mi R-126-5p overexpression reduced the luciferase activity of the WT-BCAR3 3?-UTR(P < 0.01)but not that of the Mut-BCAR3 3?-UTR construct.To determine whether BCAR3 was regulated by mi R-126-5p,NESCs were transfected with mi R-126-5p antagomir and ESCs were transfected with mi R-126-5p agomir,we found that the effect of mi R-126-5p downregulation on promoting NESC proliferation,migration and invasion was partially reversed by BCAR3 silencing(P<0.05).Upregulation of BCAR3 in mi R-126-5p stably overexpressed ESCs modestly reversed the repressive effect of mi R-126-5p on ESC migration and invasion(P<0.05).Conclusion: 1.Upregulated BCAR3 and downregulated mi R-126-5p were proved in eutopic and ectopic endometrium in EM,which were negatively correlated.In ectopic endometrium,vimentin up-regulation and E-cadherin down-regulation indicated that EMT could play a role in the development and progression of EM.2.Estrogen promoted EMT in the ectopic endometrium of endometriosis,while BCAR3 had no effect on EMT but could synergized with estrogen increasing the expression and excretion of fibronectin and fastening cell shape changing.3.BCAR3 overexpression promotes the proliferation,migration,and invasion of ESCs.The expression and function of BCAR3 were directly regulated by mi R-126-5p.Abnormal cell functions due to aberrant expression of mi R-126-5p-BCAR3 might be one of the molecular mechanism of EM development.