| Objective: Ovarian epithelial malignant tumor is the most common histological type of ovarian malignant tumor,and its mortality rate ranks the first among all kinds of gynecological malignant tumors.The reason for the high mortality rate of ovarian cancer is that it is not easy to detect in the early stage,and most cases are diagnosed in the late stage.However,there is a lack of effective treatment for advanced cases,and they have a high recurrence rate.Therefore,it is necessary to explore new valuable biomolecular markers to help improve the accuracy of diagnosis and reflect the progression of the disease before and after treatment,so as to improve the prognosis of patients.The purpose of this study was to detect the expression of BMPER in epithelial ovarian tumor tissues and analyze its relationship with the clinicopathological parameters and prognosis of ovarian cancer patients on the basis of gene microarray screening.In order to observe the effect of differential expression of BMPER on the malignant biological behavior of ovarian cancer cells,models of knockdown and overexpression of BMPER were constructed.The upstream transcription factors that regulate BMPER are predicted and verified to clarify the way of transcriptional regulation.To find the related downstream signaling pathways involved in BMPER and explore the molecular mechanism by which BMPER affects the malignant biological behavior of ovarian cancer cells,which provides new ideas and theoretical basis for molecular targeted therapy and condition monitoring of ovarian cancer.Methods: BMPER expression in 94 ovarian epithelial malignant tumors,24 ovarian epithelial borderline tumors,23 ovarian epithelial benign tumors and 30 normal ovarian tissues was detected by immunohistochemistry.Its relationship with clinicopathological parameters and prognosis was analysed.Four ovarian cancer cell lines,CAOV3,OVCAR3,SKOV3 and ES-2,were selected for cell biological behavior experiments.The expression levels of BMPER in four ovarian cancer cells were detected.Since BMPER expression was relatively high in CAOV3 and OVCAR3 cells,BMPER expression in the CAOV3 and OVCAR3 ovarian cancer cell lines was inhibited by RNA interference,and BMPER was overexpressed in ES-2 and SKOV3 cells transfected with lentivirus,and the transfection effect was verified by real-time PCR and Western blot.Changes in proliferation,migration,invasion and apoptosis in ovarian cancer cells were detected by MTT,wound healing,Transwell and flow cytometry assays as appropriate after interference and overexpression of BMPER.Western blot assay was used to detect the expression of proteins ERK1/2,p-ERK1/2,MEK1/2 and p-MEK1/2 in the MAPK signalling pathway after transfection,and the changes of molecular markers PCNA,Bax,bcl-2,MMP2 and MMP9 of ovarian cancer cell proliferation,apoptosis and migration were also detected.Subsequently,changes in the biological behavior of BMPER overexpression cells after addition of the MAPK pathway inhibitor PD98059 were detected.In the previous work,the bioinformation website PROMO database and JASPAR database were used to screen,and it was predicted that the upstream transcription factor FOXP3 might be a potential target for regulating BMPER.In this study,the binding of FOXP3 to BMPER promoter was verified by chromatin immunoprecipitation.On this basis,in order to clarify the specific regulatory effect of FOXP3 on BMPER,FOXP3 siRNA was used to transfect ovarian cancer cells CAOV3 and OVCAR3.After transfection,the changes in FOXP3 and BMPER expression were detected by Real-time PCR and Western blot,and the molecular mechanism of BMPER affecting the malignant biological behavior of ovarian cancer cells was studied in detail.Results: BMPER is mainly stained by cytoplasm and intercellular space,and a small number of nuclei are coloured with brown or brownish yellow granules.The positive rate of BMPER in ovarian epithelial malignant tumors(90.43%)was significantly higher than that in ovarian epithelial borderline tumors(73.91%),ovarian epithelial benign tumors(41.67%)and normal ovarian tissue(30.00%)(all P < 0.01).Meanwhile,the rate of positive BMPER expression in ovarian epithelial borderline tumors was higher than that in ovarian epithelial benign tumors and normal ovarian tissues(P<0.05).High BMPER expression was significantly associated with lymph node metastasis,and the proportion of patients with high BMPER expression in the lymph node metastasis positive group(86.96%)was higher than that in the lymph node metastasis negative group(64.29%)(P<0.05).Kaplan-Meier survival analysis showed that the survival rate of patients with high BMPER expression was significantly lower than that of patients with low BMPER expression.Cox regression analysis showed that high BMPER expression was an independent risk factor for poor prognosis in patients(P=0.026).BMPER was expressed in four ovarian cancer cell lines CAOV3,OVCAR3,SKOV3 and ES-2.The mRNA and protein expression levels of BMPER in CAOV3 and OVCAR3 cells were higher than those in SKOV3 and ES-2 cells,with ES-2 cells exhibiting the lowest levels of BMPER expression.The results of cell biology experiments after differential expression of BMPER showed that the proliferation,invasion and migration abilities of cells transfected with BMPER siRNA were significantly reduced compared with those of the control group,while the apoptosis rate increased.After overexpression of BMPER,the proliferation,invasion and migration of ovarian cancer cells were increased,and apoptosis was decreased.Meanwhile,compared with the control group,the siRNA transfection group had decreased expression of PCNA,decreased the expression of Bcl-2,increased the expression of the pro-apoptotic protein Bax,and decreased the expression of MMP2 and MMP9.After overexpression of BMPER,the expression of PCNA,bcl-2,MMP2 and MMP9 increased,and the expression of Bax decreased.The results were consistent with the results of cell biology experiments.After BMPER expression was down-regulated,the proportion of p-ERK / ERK and p-MEK / MEK proteins in the MAPK signaling pathway decreased.After overexpression of BMPER,the proportion of p-ERK / ERK and p-MEK / MEK proteins increased.When MAPK inhibitor PD98059 was added,the proliferation,migration and invasion of ovarian cancer cells decreased,while apoptosis increased.The results of ChIP experiments show that the transcription factor FOXP3 can bind to the-1481bp-1475 bp and-988bp--982 bp sites of the BMPER gene promoter region.After transfection of CAOV3 and OVCAR3 cells with FOXP3 siRNA,Real-time PCR and Western blot showed that FOXP3 mRNA and protein expression decreased,while BMPER mRNA and protein expression increased,indicating that FOXP3 has a negative regulatory effect on BMPER expression.Conclusions: 1.The positive expression rate of BMPER in ovarian epithelial malignant tumor group was significantly higher than that of borderline ovarian tumor group,benign ovarian tumor group and normal ovarian tissue.The prognosis of ovarian cancer patients with high expression of BMPER is worse.The high expression of BMPER is an independent risk factor for the prognosis of epithelial ovarian cancer.2.BMPER is expressed in ovarian cancer cell lines CAOV3,OVCAR3,SKOV3,and ES-2.Among them,the expression is higher in CAOV3,OVCAR3 cells,and relatively lower in ES-2 and SKOV3 cells.BMPER can promote the migration,invasion,proliferation and inhibit the apoptosis of ovarian cancer cells.3.BMPER affects the malignant biological behavior of ovarian cancer cells through MAPK signaling pathway.In ovarian cancer cell lines,the transcription factor FOXP3 can bind to the BMPER promoter and negatively regulate the expression of BMPER. |
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