| PART1The expression of EMX2in endometrial cancer and itsassociations with patients’clinicopathological parametersObjective: To investigate the expression of EMX2in endometrial cancer tissues and immotalizedhuman endometrial cancer cell lines.Methods: Immnuohistochemical staining was performed to detect the protein of EMX2in normalendometrium (n=25) and endometrial cancer tissues (n=122). Real-time PCR and western blotwere used to investigate the Mrna and protein of EMX2in endometrial cancer cell lines. Thesoftware SPSS16.0was used for the analysis about EMX2expression and patients’clinicopathological parametersResults：(1) Compared with normal endometrium, EMX2protein expression was lost in48.4%(59/122)endometrial cancer tissues (P <0.001), which was correlated with late tumor stage (P=0.023)、advanced grade (P=0.016) and deep myometrial invasion(P=0.04); however, loss of EMX2wasnot related to age, lymph vascular space invasion, lymphatic node metastasis or ER/PR status.(2) In the normal endometrium, the EMX2protein mainly localized in nuclear, and occasionally incytoplasm. We did not detect any alteraions of EMX2protein localization in the endometrialcancer tissues.(3) The mRNA and protein of EMX2were abundant in KLE、Ishikawa cells, but rarely inRL95-2、AN3CA and SPEC-2.Conclusion: EMX2was frequently lost in endometrial cancer and correlated with tumor progression, indicating that EMX2might be an important factor invovled in the development andprogression of endometrial cancer.PART2The mechanism underlying EMX2silence in endometrialcancerObjective: To determine the mechanism underlying EMX2silence in endometrial cancer.Methods: The frequency of EMX2promoter hypermethylation was detected using methylationspecific PCR (MSP) and DNA sequencing in endometrial cancer cell lines（n=4）, endometrialcancer tissues（n=79）and normal endometrium（n=22）. Real-Time PCR and western blot wereused to investigate the mRNA and protein expression of EMX2after the treatment of5-AZA-dC(a commonly used demethylating agent).Results:(1) In normal endometrium and endometrial cancer tissues, the frequency of EMX2promoterhypermethylation were4.5%（1/22） and39.2%（32/79， P <0.001）. And promoterhypermethylation was significantly correlated with the loss of EMX2expression (P <0.001).(2) RL95-2but not the other four cell lines presented EMX2promoter hypermethylation; with thetreatment of1μM5-AZA-dC for12h, both the mRNA and protein of EMX2were significantlyupregualted. This phenomenon did not occur in cells without hypermethylated EMX2.Conclusions: Promoter hypermethylation is a major mechanism for the loss of EMX2inendometrial cancer. These data implied that EMX2gene might be a tumor suppressor inendometrial cancer. PART3The affects of EMX2on the biological behaviors ofendometrial cancer cellsObjective: To explore the affects of EMX2on the biological behaviors of endometrial cancercells.Methods: The expression of EMX2was upregulated by transient transfection ofpcDNA3.1-EMX2in RL95-2cells, and downregulated by siRNA-EMX2in KLE cells. functionsof EMX2were investigated by MTT, flow cytometry, wound healing, transwell and colonyformation assay. RL95-2cells with stably EMX2overexpression was established by G418selection, and this cell line was used for the tumor xenografts growth assay.Resutls：(1) In RL95-2cells, overexpression of EMX2suppressed cellular proliferation (P=0.002forMTT and P=0.03for colony formation assay), migration (P=0.018) and invasion (P=0.039),and led to G1phase arrest.(2) In KLE cells, knockdown of EMX2promoted cellular proliferation (P=0.025for MTT and P=0.011for colony formation assay), migration (P=0.026) and invasion (P=0.019), andaccelerated cell cycle.Consistently, in the endometrial cancer mouse model, EMX2also significantly inhibited tumorgrowth (P=0.005) and tumor weights (P=0.023).Conclusions: In vitro and in vivo experiments demonstrated that EMX2could suppress tumorgrowth and progression of endometrial cancer.PART4The antitumor functions of EMX2were mediated by Wnt signal pathwayObjective: To explore the molecule mechanisms underlying EMX2-Wnt pathway in endometrialcancer.Methods: The expression of EMX2was upregulated by transient transfection ofpcDNA3.1-EMX2in RL95-2cells, and downregulated by siRNA-EMX2in KLE cells. Then realtime PCR and western blot were performed to investigate the alterations of β-catenin and cyclinD1.The Wnt signal inhibitor XAV-939or activator LiCl was used to block or reactivate the Wnt signalpathway, and the antitumor functions of EMX2were measured by MTT assay and cell cycleanalysis.Results:(1) In RL95-2cells, overexpression of EMX2significantly suppressed the mRNA and proteinlevel of β-catenin; LiCl activitated the Wnt/β-catenin signal and eliminated the suppressive effectsof EMX2on cell proliferation and cell cycle progression, which also restored the expression ofcyclin D1and CDK4.(2) Meanwhile, in KLE cells, downregulation of EMX2restored the expression of β-catenin.Treatment with XAV-939blocked the Wnt/β-catenin pathway, which suppressed cellularproliferation, cell cycle progression, and downregulated the expression of cyclin D1and CDK4.Conclusion: Wnt/β-catenin signal is a key pathway for EMX2gene in endometrial cancer. |
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