| Objective:Epithelial ovarian cancer has the highest mortality rate among gynecological tumors.70% of patients are diagnosed at an advanced stage,which is a malignant tumor that is difficult to diagnose early.Although ovarian cancer has been greatly improved in treatment,the 5-year survival rate of ovarian cancer is still hovering between 25% and 45% due to recurrence,metastasis,drug resistance,and immune escape.Finding efficient and specific tumor markers and effective drug targets are very important for the diagnosis and treatment of ovarian cancer.Human epididymis protein 4(HE4)is an extracellular secreted protein,named for its specific expression in epididymal epithelial cells.In 1999,Schummer et al.first discovered that HE4 was highly expressed in ovarian cancer through genomic comparison.Researchers conducted a meta-analysis on 2607 items and found that HE4 has a higher sensitivity and diagnostic value in the diagnosis of ovarian cancer.Specificity,its positive likelihood ratio and negative likelihood ratio are better than CA125,and the positive rate of HE4 in benign diseases is lower than CA125,which indicates that it has more advantages in the diagnosis of ovarian cancer.It was designated as a serum marker of ovarian cancer in 2003;it was approved by the FDA in 2009 to monitor the recurrence and progression of epithelial ovarian cancer.HE4 is a secreted protein,so the current research is mostly limited to reports of serum HE4 in the diagnosis and monitoring of epithelial ovarian cancer,but there are few studies on the function and mechanism of HE4.Our team has used clinical specimens and ovarian cancer cell lines to show that HE4 is highly expressed in ovarian cancer tissues and serum,and has proven that HE4 and Annexin A2 are interacting proteins.The mutual combination of the two activates MAPK and FOCAL adhesion.We used the yeast two-hybrid method with HE4 as the bait,screened in the c DNA library and performed gene sequencing to obtain the YWHAE protein.YWHAE protein(also known as 14-3-3ε protein),which is a tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activating protein,belongs to a member of the 14-3-3 protein family.This family is a class of highly conserved proteins that play a bridge role in protein interactions and bind extensively to other proteins such as kinases,phosphatases,transmembrane receptors,and transcription factors.As an intermediate protein,YWHAE can bind to a variety of target proteins to regulate tumor cell growth,migration,metastasis,cell cycle,apoptosis,and participate in signal transduction pathways,which in turn affects tumor development.YWHAE protein has been shown to be highly expressed in intestinal cancer,liver cancer,lung cancer,kidney cancer,and esophageal cancer in tumor-related studies,but the specific mechanism remains unclear.With the development of gene detection technology and bioinformatics,although there have been reports of differential expression of YWHAE gene in ovarian cancer before and after chemotherapy and in patients with abnormal glucose metabolism,it is expressed in human ovarian epithelial tumor tissues.Experimental studies on the situation and mechanism of action have not been reported.This study explores the relationship between YWHAE and HE4,and the clinical significance of YWHAE expression in ovarian tissues based on the previous work.This study constructs a YWHAE differentially expressed cell model and explores the effect of YWHAE on the malignant biological behavior of ovarian cancer cells.Exploration the downstream pathway activated by YWHAE provides a theoretical basis for further exploring the occurrence and development mechanism of ovarian cancer and finding new therapeutic targets for ovarian cancer.Methods:1.In this study,the interaction between YWHAE and HE4 was first detected by co-immunoprecipitation,and the cell localization relationship between YWHAE and HE4 was detected by fluorescence confocal method.A total of 163 paraffin specimens were surgically removed from Shengjing Hospital’s Obstetrics and Gynecology Department from 2008 to 2012,including 16 cases in the normal group,18 cases in the benign group,24 cases in the borderline group,and 105 cases in the malignant group.Immunohistochemical method is used to detect the expression level of YWHAE protein,and analyzed the relationship between clinical pathological parameters and the YWHAE expression.The effect of YWHAE expression on the prognosis of the patients was analyzed after analyzing survival the 105 patients with epithelial ovarian cancer.2.Use lentivirus to construct YWHAE stable and highly expressing cell lines OVCAR3-YWHAE-H and its control cell lines OVCAR3-YWHAE-Mock,A2780-YWHAE-H and its control cell line A2780-YWHAE-Mock;use small interfering RNAs respectively The YWHAE low-expressing cell line CAOV3-YWHAE-L and its control cell lines CAOV3-YWHAE-Mock,ES2-YWHAE-H and its control cell line ES2-YWHAE-Mock were constructed.Transwell test was used to detect the invasion and EMT of cells before and after differential expression of YWHAE,scratch test was used to detect cell migration ability,MTT was used to detect cell proliferation ability,and flow cytometry was used to detect changes in cell cycle and apoptosis.Immunohistochemical detection of p-AKT and p-ERK protein expression in nude mice tumor tissue samples).3.The Western Blot method was used to detect changes in PI3 K and MAPK signal pathway proteins with a differential expression of YWHAE.Transwell test,scratch test,and MTT test were used to detect cell invasion,migration,and proliferation biological behaviors before and after the addition of corresponding pathway inhibitors.Results:1.This study demonstrated that YWHAE and HE4 interacted with each other and co-localized by co-immunoprecipitation and fluorescence confocal methods.Immunohistochemical experiments proved that the positive expression rate and strong positive rate of YWHAE in the malignant ovarian group were significantly higher than those in the borderline,benign and normal groups.Analysis of clinical pathological parameters suggested that the high expression rate of YWHAE in patients with FIGO III-IV ovarian cancer It is significantly higher than that in patients with FIGO I-II stage ovarian cancer,and patients with high expression of YWHAE have poor prognosis.High expression of YWHAE and FIGO staging are independent risk factors for prognosis of patients with ovarian cancer.Correlation analysis suggests that the expression of YWHAE is positive for HE4 Related.2.The invasion and migration capabilities of tumor cells with high expression of YWHAE were significantly enhanced,and with different expression of YWHAE,the invasion and migration capabilities of tumor cells were significantly weakened.In tumor cells with high expression of YWHAE protein,the apoptotic cells proportion decreased,the tumor cell proliferation ability in vitro was significantly enhanced and the proportion of G2/M phase cells increased significantly.In vivo experiments(tumor formation in nude mice)found that,the volume of xenografts produced by cells in the YWHAE overexpression group was significantly larger.It was proved that YWHAE can enhance malignant biological behaviors such as tumor cell invasion,migration,proliferation,and inhibition of apoptosis.Nude tumor formation experiments proved,that the volume and quality of xenograft tumors produced by the YWHAE high-expressing group were significantly larger;the immunohistochemical results of the nude mouse tumor tissue showed,the strong expression of p-AKT and p-ERK proteins are much higher.3.The phosphorylation of PI3 K / AKT pathway node proteins(PI3K/AKT/m-TOR)and MAPK pathway node proteins(MEK/ERK)in YWHAE over-expressing cell lines was significantly increased.The ability of ovarian cancer cells over-expressing YWHAE to invade,migrate and proliferate was reduced,after addition of pathway inhibitors(PI3K and MAPK).Conclusion:1.This study proves that YWHAE and HE4 interact and co-localize.The expression of YWHAE was significantly increased in ovarian cancer tissues,it was an independent risk factor for the prognosis of ovarian cancer,and it was positively correlated with the expression of HE4.2.YWHAE can promote the invasion,migration,proliferation of ovarian cancer cell lines,and inhibit malignant biological behaviors such as apoptosis.3.YWHAE can promote the invasion,migration and proliferation through PI3 K / AKT and MAPK pathways of epithelial ovarian cancer cells. |
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