Mechanism Of DEC1 Affecting The Biological Behavior Of Epithelia Ovarian Cancer Cells By Regulating The Wnt/β-catenin Signaling Pathway

Background and Objective:Ovarian cancer(OC)is one of the most common gynecologic malignancies.The most common pathological type of ovarian cancer is epithelial ovarian cancer(EOC),accounting for 80-90%.Compared with endometrial carcinoma,cervical carcinoma and vulvar carcinoma,the mortality rate of OC ranks first in female genital tract malignant tumors,and the incidence rate increases year by year,which seriously threatens the life and health of women.According to the American Cancer Society,there were approximately 22,530 new cases and 13,980 deaths in the United States in2019.Anatomically,the ovary is located in the deep pelvic cavity,with no obvious clinical manifestation in the early stage,and in addition,there is a lack of early screening measures,making it difficult for the patient to be diagnosed in the early stage.Clinically,about 70% of patients present with abdominal distension,ascites and abdominal pain for the first time,and the diagnosis is in the advanced stage.The main clinical characteristics of OC include concealed onset,late detection,high malignancy,easy metastasis and poor prognosis.The 5-year survival rate can be over 75% in patients with early OC and only 30-40% in patients with advanced OC.Cytoreductive surgery of ovarian tumors and platinum-based chemotherapy as well as PARP inhibitors are standardized treatment modalities for patients with advanced OC.With the improvement of diagnosis and treatment level,the five-year survival rate of OC patients increased from 36% in 1975 to 47% in 2014.However,about 70% of patients with advanced OC still relapse within the first 2-3 years after treatment,with continuous recurrence and chemotherapy,eventually leading to tumor resistance.Therefore,for the current situation of difficult to detect,high recurrence,difficult to treat and poor prognosis of the ovary,it is urgent to develop new treatment strategies to improve the prognosis of OC patients.Differential Embryonic Cartilage Development Gene 1(DEC1),belonging to the BHLH transcription factor family,is expressed to varying degrees in most organs and tissues of the human body.DEC1 plays an important role in a variety of physiological functions of the body,involving cell differentiation,regulation of cell cycle and circadian rhythm,hypoxia,stress response,and other processes.In recent years,it has been found that DEC1 is abnormally expressed in many tumors and is related to tumor growth and apoptosis.For example,DEC1 has a positive anti-apoptotic effect on hypoxia-induced growth of gastric cancer cells.DEC1 negatively affected hypoxia-induced E-cadherin expression in Hep G2 cells.Silencing DEC1 inhibits the proliferation of breast cancer by inducing S-stage cell cycle arrest.The Wnt/β-catenin signaling pathway is one of the most important signaling pathways in cells,which plays an important role in regulating the normal growth,movement,differentiation and tumorigenesis of cells.As a key molecule of Wnt/β-catenin,β-catenin was accumulated in the cytoplasm and transferred to the nucleus,where it activated the downstream signaling molecules.The existing studies have shown that the abnormal activation of wnt/β-catenin pathway is closely related to the occurrence and development of many tumors.For example,Wang et al.found that after the down-regulation of gene expression in H1299 cells,the wnt/β-catenin signaling pathway was inhibited,and the expression level of GSK3β was significantly increased,while the expressions of p-GSK3β,nuclear-β-catenin,cyclin,c-Myc,and MMP-7 were significantly decreased,resulting in the decreased proliferation and invasion abilities of H1299 cells.Studies have shown that in normal colorectal tissues,the wnt signaling pathway regulates the balance,proliferation and differentiation of intestinal stem cells.Other studies have shown that the wnt pathway in OC cells is activated,which promotes β-catenin to enter the nucleus and act on TCF and LEF transcription factors to induce the growth,differentiation and metastasis of OC stem cells.The above studies have shown that the abnormal expression of DEC1 in a variety of other tumors leads to tumor cell proliferation and invasion,however,the possible effect and potential mechanism of DEC1 on ovarian cancer are still unclear.This study aimed to explore the functions and mechanisms of DEC1 in EOC.We speculated that the down-regulation of DEC1 could significantly inhibit the proliferation,migration,and invasion of EOC cells and induce apoptosis through the wnt/β-catenin signaling pathway.Our results will provide new insights into the biological functions and potential mechanisms of DEC1 in EOC and new therapeutic targets for the diagnosis or treatment of EOC.Methods:1.The m RNA and protein expression levels of DEC1 in 106 epithelial ovarian cancer tissues and adjacent tissues were detected by q RT-PCR,immunohistochemistry and Western blot,and the correlation between the expression of DEC1 and the overall survival(OS)of patients was analyzed by Kaplan-Meier method.2.DEC1 protein and m RNA expressions in human normal ovarian epithelial cell line(IOSE80)and epithelial ovarian cancer cell lines(SKOV3 and OVCAR3)were detected by Western blot and q RT-PCR assays.The SKOV3 and OVCAR3 cell lines were transfected with the DEC1 interfering plasmid to construct a sh DEC1 stably transfected cell line,and the transfection effect was detected by Western blot and q RT-PCR.3.Observe the changes of proliferation ability of sh DEC1 epithelial ovarian cancer cells after down-regulating the expression of DEC1 in epithelial ovarian cancer cells through CCK-8 and Ed U experiments;4.TUNEL assay was used to observe the changes of apoptotic level of sh DEC1 ovarian cancer cells after down-regulating the expression of DEC1 in epithelial ovarian cancer cells,and Western blot was used to detect the changes of expression levels of apoptosis-related proteins(Caspase-3,Cleaved-caspase-3,PARP and Cleaved-PARP).5.The changes of invasion and migration abilities of sh DEC1 epithelial ovarian cancer cells after down-regulating the expression of DEC1 in ovarian cancer cells were observed by Transwell and scratch experiments,and the expression levels of EMT-related proteins(E-cadherin,Vimentin and α-SMA)were detected by Western blot.6.Western blot was used to detect the changes in the expression levels of Wnt/β-catenin signaling pathway key proteins(p-GSK3β and β-catenin)in epithelial ovarian cancer cells after the expression of DEC1 was down-regulated.The total protein and nucleoprotein expressions of β-catenin and TCF4 transcriptional activity in epithelial ovarian cancer cells were detected after down-regulation of DEC1 expression.Western blot and protein immunoprecipitation assay were used to detect the changes of β-catenin protein ubiquitination after the down-regulation of DEC1 expression in epithelial ovarian cancer cell lines.Results:1.The m RNA level of DEC1 in epithelial ovarian cancer tissue is significantly higher than that in the paracancerous tissue(P < 0.01);The protein expression level of DEC1 was significantly higher than that of adjacent tissues(P <0.01).Patients with high-expression DEC1 EOC had shorter overall survival(OS)than patients with low-expression DEC1 EOC(P < 0.05).2.The protein expression and m RNA levels of DEC1 in epithelial ovarian cancer cells(SKOV3 and OVCAR3)are significantly higher than those in normal human ovarian epithelial cells(IOSE80);After transfection of the DEC1 interfering plasmid,the protein expression and m RNA levels of DEC1 in epithelial ovarian cancer SKOV3 and OVCAR3 cells were significantly down-regulated.3.Down-regulating the expression of DEC1 results in significant inhibition of cell proliferation in epithelial ovarian cancer SKOV3 and OVCAR3 cells.4.The apoptotic level is significantly increased after the expression of DEC1 is down-regulated in epithelial ovarian cancer cells;The detection of expression of apoptosis-related proteins revealed a decrease in Caspase-3 protein expression,an increase in Cleaved-caspase-3 protein expression,a decrease in PARP protein expression,and an increase in Cleaved-PARP protein.5.The down-regulated expression of DEC1 in epithelial ovarian cancer SKOV3 and OVCAR3 cells results in significant inhibition of cell invasion and migration.The detection of EMT-related protein expression showed that the expression of E-cadherin protein was significantly increased,while the expression of Vimentin andα-SMA proteins were decreased.6.Down-regulating the expression of DEC1 in epithelial ovarian cancer cells results in unchanged expression of Wnt/β-catenin signaling pathway key protein GSK3β,decreased expression of p-GSK3β protein and decreased expression ofβ-catenin in the cells.Western blot and protein immunoprecipitation assay detected the increase of β-catenin ubiquitination and the down-regulation of β-catenin protein expression in epithelial ovarian cancer cells(SKOV3 and OVCAR-3)after down-regulation of DEC1 expression.Conclusion:In summary,the above results have shown that 1.DEC1 expression in both tissues and cells of epithelial ovarian cancer is significantly increased,and it is closely related to the poor prognosis of ovarian cancer patients;2.Down-regulating the expression of DEC1 in epithelial ovarian cancer cells can significantly inhibit cell proliferation,invasion and migration capacity and induce apoptosis by regulating the wnt/β-catenin signaling pathway.Our results provide new ideas for studying the biological function and potential mechanism of DEC1 in OC and new therapeutic targets for the diagnosis and treatment of OC.