The Effect And Mechanism Of Tumor Suppressor PDCD4 In Endometriosis

Objective:Endometriosis(EM)is a kind of common,estrogen-dependent disease that occurrs in 10%-15%women of reproductive-age.It is defined as the presence of uterine endometrial glands and stromas outside of the uterine cavity,including pelvis,upper abdomen,lungs,even central nervous system.The predominant sites of endometriosis were the ovaries(96.4%),followed by the soft tissue(2.8%),gastrointestinal tract(0.3%),and urinary tract(0.2%).Endometriosis could result in chronic pelvic pain,nearly 50%women with endometriosis have chronic pelvic pain,specially during menstruation.In addition,endometriosis is also the most important cause of infertility,30-50%women with endometriosis have infertility.Although endometriosis is a benign disease,previous reports suggest that it shares many similar features with cancers,such as cell migration,invasion and unrestrained growth.Epithelial to mesenchymal transition-like(EMT)and EMT-like processes might be also related to the pathogenesis of pelvic endometriosis.It has been reported that 10%-15%of endometriosis cases are found to be more aggressive and tend to invade deep into the affected tissues and organs,producing more severe clinical symptoms,and deep infiltrating endometriosis usually respond bad to hormonal suppressive therapy.The main theory for the formation of ectopic endometrium was retrograde menstruation and implantation.The retrograde menstruation is a physiologic process that takes place almost in all menstruation cycles of reproductive-age women,however,the morbidity of endometriosis is only 10-15%.Now,more and more studies show that the primary defect in endometriosis can be located in the eutopic endometrium.It has been known that some genes are expressed differentially in eutopic endometrium of endometriosis patients compared with control endometrium.The cellular and molecular characteristics of eutopic endometrium in patients with endometriosis have not been elucidated.It is required to further study the mechanism of endometrial cell migration and invasion,and develop new and effective methods for diagnosis and treatment of endometriosis.Programmed cell death 4(PDCD4)was initially identified as a protein induced by apoptotic stimuli.Subsequent studies showed that PDCD4 could be acted as tumor suppressor,and play important roles by inhibiting protein translation and gene transcription.PDCD4 mRNA and protein were significantly downregulated in various human cancers such as hepatocellular carcinoma,glioma,breast carcinoma,gastric cancer and ovarian cancer.PDCD4 overexpression can induce tumor cell to apoptosis,cell cycle arrest,increase sensitivity of tumor cells to antitumor drugs,and inhibit tumor cells migration and invasion,as well as angiogenesis.Besides,PDCD4 was also involved in obesity,adipose tissue inflammation,atherosclerosis and LPS/D-GaIN induced acute injury as an inflammatory factor.Considering the critical functions of PDCD4 in malignant tumor and inflammation,the possible roles of PDCD4 in endometriosis remain to be clarified.In the present study,we detected the expression of PDCD4 in endometrium of women with or without endometriosis and analyzed the effects of PDCD4 on the biological behaviors of endometrial cells.Materials and Methods:1.The expression of PDCD4 in control endometrium and eutopic and ectopic endometrium of endometriosis patientsWe firstly detected the expression of PDCD4 mRNA and protein in the eutopic endometrium of endometriosis patients(26)and control endometrium(35)by qRT-PCR and western blot.Next,we further detected the expression of PDCD4 protein in eutopic or ectopic endometrium of endometriosis patients and control endometrium by IHC.2.The effect of progesterone or estrogen on expression of PDCD4 in endometrial cellsWe treated the endometrial cells with different concentration of estrogen(0.1nM,1nM,10nM,100nM and 1000nM)or progesterone(0.01uM,0.1uM,1uM,10uM and 20uM)and detected the expression of PDCD4.Then,the endometrial cells were treated with progesterone(10uM)for 6h,12h,24h,36h and 48h,followed by expression analysis of PDCD4.3.The effect of PDCD4 on the proliferation,migration and invasion of endometrial cellsTo explore the effect of PDCD4 on the growth,migration and invasion ability of endometrial cells,we firstly compared the difference in PDCD4 expession betweenHEC-1-A cells or Ishikawa cells transfected PDCD4 plasmid and control cells,as well as Ishikawa cells transfected PDCD4 sprecific siRNA-1 or siRNA-2 and control cells.CCK8,colony formation and transwell assay were used to detect the effect of PDCD4 on the proliferation,migration and invasion of endometrial cells.4.The effect of PDCD4 on the cell cycle and apoptosis of endometrial cellsIn order to investigate the mechanism by which PDCD4 inhibited the cell viability and growth,we determined the effect of PDCD4 on the cell cycle and apoptosis using HEC-1-A cells transfected PDCD4 plasmid and Ishikawa cells transfected PDCD4 sprecific siRNA-1 or siRNA-2.Flow cytometry was used to determine the effect of PDCD4 on the cell cycle and apoptosis of endometrial cells.5.The effect of PDCD4 on the autophagy of endometrial cellsIn order to investigate the mechanism by which PDCD4 inhibits the proliferation of endometrial cells.We detected the expression of LC3B-II after PDCD4 overexpression or knockdown by western blot.6.The effect of PDCD4 on the signal pathway in endometrial cellsIn order to investigate the mechanism by which PDCD4 inhibits the migration and invasion of endometrial cells.We detected the expression of P65,p-P65,MMP2 and MMP9 after PDCD4 overexpression or knockdown by western blot.7.The effect and mechanism of PDCD4 on the proliferation and migation of primary endometrial glandular epithelial cells and stomal cellsTo further determine the effect of PDCD4 on the proliferation and migration of endometrial cells,primary endometrial cells including endometrial glandular epithelial cells and endometrial stromal cells were isolated and identified from secretory phase of control endometrium.CCK8 and transwell assay were used to detect the effect of PDCD4 on the proliferation and migration of primary endometrial cells.Western blot was used to detect the effect of PDCD4 on the expression of P65,p-P65,MMP2 and MMP9 in primary endometrial cells.Results:1.PDCD4 expression was downregulated in the eutopic and.ectopic endometrium of endometriosis patients compared with control endometriumThe results from qRT-PCR and western blot showed that PDCD4 mRNA and protein levels in the eutopic endometrium of endometriosis patients were significantly lower than those in control endometrium.The results from IHC showed that PDCD4 protein was mainly located in the cytoplasma of glandular epithelium,while no obvious positive staining could be observed in endometrial stromal cells.PDCD4 protein levels in the eutopic and ectopic endometrium of endometriosis patients were obviously downregulated compared with control endometrium.In addition,the staining index of PDCD4 in the proliferative phase was significantly higher than that in the secretory phase of control endometrium and the proliferative phase of eutopic endometrium,as well as ectopic endometrium.2.Progesterone suppresses PDCD4 expression in endometrial cells,but estrogen has no evident effect on PDCD4 expressionWe treated the endometrial cells with different concentration of estrogen or progesterone and detected the expression of PDCD4.Then,the endometrial cells were treated with progesterone(10uM)for 6h,12h,24h,36h and 48h,followed by expression analysis of PDCD4.Western blot data showed that Progesterone suppresses PDCD4 expression in endometrial cells,but estrogen has no evident effect on PDCD4 expression.3.PDCD4 suppressed the growth and colony forming ability of endometrial cellsThe results confirmed that HEC-l-A cells or Ishikawa cells transfected PDCD4 plasmid grew significantly more slowly than the control cells,and the colony number and size of HEC-l-A cells or Ishikawa cells transfected PDCD4 plasmid were significantly reduced compared with the control group.In contrast,the living cell number,colony number and size were obviously increased in Ishikawa cells transfected PDCD4 sprecific siRNA-1 or siRNA-2 compared with the control cells.4.PDCD4 had no effect on cell cycle and apoptosis in endometrial cellsWe determined the effect of PDCD4 on the cell cycle and apoptosis using HEC-1-A cells transfected PDCD4 plasmid and Ishikawa cells transfected PDCD4 sprecificsiRNA-1 or siRNA-2.The results revealed that PDCD4 overexpression and knockdown had no significant effect on the cell cycle and apoptosis of endometrial cells.5.PDCD4 inhibits autophagy in endometrial cellsPDCD4 overexpression significantly decreased the levels of LC3B-Ⅱ.In contrast,the silence of PDCD4 increased the expression of LC3B-Ⅱ.6.PDCD4 repressed the migration and invasion of endometrial cellsThe results from transwell migration assay showed that PDCD4 overexpression in HEC-l-A or Ishikawa cells resulted in significant reduction in the number of cells passing through the chambers compared with the control groups.Inversely,the Ishikawa cells transfected with PDCD4 specific siRNA migrated into the lower surface of the transwell membrane were increased.We also performed matrigel invasion assay using Ishikawa cells and HEC-l-A cells.The number of cells passing through the chambers in the PDCD4 plasmid-transfected groups was significantly smaller than that in the control groups,while PDCD4 knockdown accelerated the number of cells into the lower surface of the transwell membrane.7.PDCD4 inhibited the migration and invasion of endometrial cells by NF-κB/MMP2/MMP9 signal pathwayWe detected the expression of P65,p-P65,MMP2 and MMP9 after PDCD4 overexpression and knockdown.The results showed that the expression of p-P65,MMP2 and MMP9 was significantly downregulated after PDCD4 overexpression in HEC-1-A cells,while the levels of p-P65,MMP2 and MMP9 were increased in Ishikawa cells transfected with PDCD4 specific siRNA.8.PDCD4 inhibited the migration and growth of primary endometrial cellsThe results from immunocytochemistry showed that stromal cells expressed vimentin and glandular epithelial cells expressed cytokeratin.We found that PDCD4 overexpression significantly suppressed cell growth and migration in primary endometrial cells.However,PDCD4 knockdown accelerated cell growth and migration of primary endometrial cells.9.PDCD4 inhibited the migration of primary endometrial cells by NF-κB/MMP2/MMP9 signal pathwayPDCD4 overexpression significantly decreased the levels of p-P65,MMP2 and MMP9.In contrast,the silence of PDCD4 increased the expression of p-P65,MMP2 and MMP9.Conclusion:1.PDCD4 expression is reduced in eutopic and ectopic endometrium of patients with endometriosis.2.Progesterone suppresses PDCD4 expression in endometrial cells,but estrogen has no evident effect on PDCD4 expression.3.PDCD4 inhibits the growth and colony formation of endometrial cells,but PDCD4 had no effect on cell cycle and apoptosis.4.PDCD4 inhibits autophagy in endometrial cells.5.PDCD4 suppresses the migration and invasion of endometrial cells mediated by NF-κB/MMP2/MMP9 signal pathway.6.PDCD4 inhibits the migration and growth of primary endometrial epithelial cells,the mechanism of inhibiting migration is related to NF-κB/MMP2/MMP9 signal pathway.Innovations and significances:1.For the first time,we demonstrate the correlation between PDCD4 and endomentrosis.We find PDCD4 expression is reduced in endomentrosis.2.PDCD4 suppresses the proliferation,migration and invasion of endometrial cells.Studies show that the primary defect in endometriosis can be located in the eutopic endometrium.3.Our findings reveal a novel approach to target the aberrant PDCD4 expression in endometriosis.