Effects Of EFEMP2 On The Invasion And Metastasis Of Endometrial Cancer

Background:Endometrial carcinoma is the second most common malignant tumor occurring in the female genital tract.Although early-stage endometrial cancer generally has a favorable outcome,up to 30%of endometrial cancer patients are diagnosed with International Federation of Gynecology and Obstetrics(FIGO)stage Ⅲ or Ⅳ,and have a relatively poor prognosis,with local or extra-pelvic metastasis,and even distant metastasis.Metastasis causes most cancer deaths,so there is an urgent requirement for exploring the mechanism of tumor invasion and metastasis to further elucidate the progression of cancers.Recently,many biomarkers that are related with the development of endometrial carcinoma have been evaluated;however,no information has been found regarding the function of EFEMP2 in endometrial cancer cell invasion and metastasis.EFEMP2(endothelial growth factor(EGF)-containing fibulin-like extracellular matrix protein 2)is critical molecules for elastic fiber assembly.Meanwhile,EFEMP2 is associated with many diseases,including cutis laxa,aortic dissection,osteoarthritis,and cancer.At present,research on the correlation between EFEMP2 and tumors is still in the initial stage.The role of EFEMP2 in endometrial cancer cell growth and metastasis remains unexplored.In order to clarify the effects of EFEMP2 on the progression of endometrial cancer and the function of EFEMP2 in endometrial cancer cell proliferation,invasion,and metastasis.This is the first study to explore EFEMP2 expression in endometrial cancer tissues and cell lines and the related mechanism.Objective:1.To investigate expression and the significance of EFEMP2 in endometrial cancer tissues and cell lines.2.To investigate the function of strongly and weakly invasive subclones after lentivious transfection.3.To investigate the mechanism of EFEMP2 in the process of invasion and metastasis of endometrial cancer cells.Methods:1.Immunohistochemical(IHC)staining was performed to determine EFEMP2 expression in normal endometrial and endometrial carcinoma tissue;2.Using the single cell cloning technique,strongly invasive subclones KLE-1、ISK-land weakly invasive subclones KLE-28、ISK-23 were obtained from KLE、ISK;3.RT-qPCR,Western blot and ICC were performed to investigate EFEMP2 expression in normal endometrial cell and weakly invasive subclones compared with endometrial carcinoma cell lines and strongly invasive subclones;4.Growth curve and soft agar assay were used to investagate the proliferation of endometrial cancer cells and subclones;5.Transwell was performed to investagate the invasion and metastasis of endometrial cancer cells and subclones;6.Using lentivirus transfection,EFEMP2 shRNA and pLVX-EFEMP2 were constructed and used to infect the strongly and weakly invasive subclones and verify thetransfection efficiencies;7.Establishing tumor xenografts in nude mice to simulate the process of endometrial cancer in vivo,and exploring the effects of EFEMP2 on cell proliferation,invasion,and migration of endometrial cancer;8.Western blot and RT-qPCR were performed to investigate the effects of EFEMP2 on the Epithelial-Mesenchymal Transition(EMT)of endometrial cancer.Results:1.EFEMP2 was down-regulated in endometrial cancer tissue and cell compared with normal endometrial tissue(P<0.05),the expression of EFEMP2 was significantly related with high stage,low differentiation and positive lymph node status of endometrial cancer(P<0.05);2.Real-time RT-PCR,Western blotting and ICC results confirmed low expression of EFEMP2 in endometrial cancer cell lines and strongly invasive subclones(P<0.05);3.The cell proliferation,invasion and migration abilities of weakly invasive subclones were significantly promoted by EFEMP2 knockdown;accordingly increased EFEMP2 could inhibit the proliferation,invasion and migration abilities of strongly invasive subclones(P<0.05);4.Tumor xenografts in nude mice:The average tumor volume and growth speed of strongly invasive subclones were much higher than weakly invasive subclones;the average tumor volume,tumor formation rate and lung metastasis of RNA interference group were much higher than EFEMP2 over expression group(P<0.05).5.Fibulin-4 upregulation increased the expression of E-cadherin,reduced the expression of N-cadherin、Vimentin、Snail‘Slug and Twist,and promoted EMT;fibulin-4 knockdown decreased the expression of E-cadherin,increased the expression of N-cadherin、Vimentin、Snail、Slug and Twist,and inhibited EMT(P<0.05).Conclusion:1.EFEMP2 may be a tumor suppressor gene of endometrial cancer,EFEMP2 may have the ability to suppress endometrial cancer cell invasion and migration.3.EFEMP2 inhibits cancer cell invasion and metastasis by preventing EMT proceeding.