The Role Of Wnt10a/Wnt10b And The Wnt/β-catenin Signaling Pathway In Endometrial Cancer

Section OneThe expression and clinical significance of Wnt10a and Wnt10b in endometrial cancerObjective:To determine the expression of WntlOa and WntlOb in different status of endometrium tissue, explore the relationship among their expression and the clinicopathologic characteristics and prognosis, dsicover the function of WntlOa and Wnt10b in the genesis and development of endometrial cancer (EC). Explore valuable data for EC nosetiology and prognosis.Methods:102EC patients hospitalized in Tianjin Medical University General Hospital from2001to2010were included in the case group. The102EC caases including95estrogen dependent (type Ⅰ) and7non-estrogen dependent (type Ⅱ), The control group included18simlpe hyperplasia,6complicated hyperplasia,30atypical,84normal endometrium(48normal hyperplasia phase and36normal secretory phase) examples were also contained. Immunohistochemistry SP method was applied to evaluate the expression of Wnt10a and Wnt10b in the tissue chips. All data were analyzed by SPSS software package (version14.0). Comparision between WntlOa and Wnt10b expression in EC、AHE、HE and NE tissue were performed using chi-squaretest. Comparisons between Wnt10a and WntlOb expression and clinicalpathological variables were used to estimate the association between Wnt10a and Wnt10b expression in EC tissues and overall patient survival. Survival curves were generated using Kaplan-Meier estimation, and the differences between curves were evaluated by log-rank tests. P values less than0.05were considered statistically significant.Results:1. The Wnt10a level of the cancerous tissues was higher than that in the normal samples, but no statistically significance was found, P>0.05(x2=2.81, P=0.25). The expression of Wnt10b in the cancerous tissues were significantly higher than that in the hyperplastic and normal examples, P<0.05(x2=8.12, P=0.02).2.The expression of WWnt10a in the endometroid cancerous tissues was significantly higher than that in other types of cancerous samples, P<0.05(x2=4.75, P=0.03).No significant differences of Wnt10a level were found among subgroups of histologic type, grade of differentiation, FIGO phase, myometrial invasion and lymphovascular metastasis, P>0.05(P=0.36, P=0.75, P=0.37, P=0.54). However, significant difference of Wnt10b level was found among the subgroups of grade of differentiation and lymphovascular metastasis and FIGO phase, P<0.05(P=0.03, P=0.02, P=0.04). No difference was found between the subgroups of myometrial invasion, P>0.05(x2=2.27, P=0.13).3. No correlationship was observed between the expression of Wnt10a and Wnt10b, P>0.05(rs=0.08, P=0.43).4. In the prognosis follow-up study, no difference was found between Wnt10a high-expression and low-expression cases of EC WntlOb gene expression was frequently up-regulated in EC, P>0.05(x2=0.027, P=0.868). However, the prognosis of WntlOb high-expression cases were much better than Wnt10b low-expression cases, P<0.05(x2=3.952, P=0.047). Conclusion:Wnt10b likely plays an important role in the development of EC. Furthermore, the prognosis of Wnt10b positive expression was better than that of negative expression. The role of Wnt10a in EC still requires further investigation.Section TwoThe study of the canonical Wnt signaling pathway in endometrial cancerObjective:To observe the role of Wnt10b on proliteration and apoptosis of endometrial cancer cell lines through Wnt/B-catenin pathway.Explore the effect of Wnt/β-catenin pathway in the genesis and development of endometrial cancer and provide theoretical foundation for the pathogenesis of endometrial cancer.Methods:Wnt10b was low-expression in AN3CA cell line, but it was high-expression in Ishikawa3-H-12cell line. Thus, we observed the effect of Wnt10b on the key factors in Wnt/β-catenin pathway including β-catenin、APC and c-myc in AN3CA cell line and Ishikawa3-H-12cell line.Cell culture, resuscitation, passage and cryopreservation were performed in sterile condition. Cell proliferation and apoptosis was performed by MTT and flow cytometry, respectively. To observe the effect of Wnt10b on the key factors of Wnt/B-catenin pathway, The mRNA leval and protein expression of β-catenin、APC and c-myc were detected using RT-PCR and Wester-blot respectively, after plasmid transfection and siRNA knockdown. All the data were analyzed by variance analysis, Fisher exact test and t test using SPSS14.0. P values less than0.05were considered statistically significant.Results:1. Impact of Wnt10b expression on ECcell proliferation:(1) The proliferation of AN3CA cells with forced Wnt10b expression was higher than that of control groups.(2) The proliferation of Ishikawa3-H-12cell in the Wnt10b -knockdown group was lower than that of control group.2. Impact of WntlOb expression on EC cell apoptosis. We observed the effect of Wnt10b on the apoptosis in AN3CA and Ishikawa3-H-12cell lines. Flow cytometric assay showed that Wnt10b expression was significantly lower than that of control groups in EC AN3CA cell lines (P<0.05).(2) The percentage of apoptosis cells in Wntl Ob-knockdown group was significantly higher than that of control groups in EC Ishikawa cell lines (P<0.01).3. Effecs of Wnt10b expression on the transcription of β-catenin. APC and c-myc:(1) WntlOb expression plasmid was transfected in AN3CA cell line. Transfection efficiency was detected by RT-PCR. We have founded that the transcription of WntlOb, β-catenin and c-myc was increased markedly, but the transcription of APC was decreased.(2) The transcription of Wnt10b,β-catenin and c-myc was decreased, but the transcription of APC was increased obviously when we knockdown Wnt10b gene in Ishikawa3-H-12cell line.4. Effects of WntlOb expression on the translation of B-catenin, APC and c-myc:(1) We detected the transfection efficiency by Western Blot after WntlOb expression plasmid had been transfected in AN3CA cell line. The results showed that the expression of APC was decreased.(2) The expression of Wnt10b, plasmid had been transfection efficiency data suggests that the enforced expression of Wnt10b, β-catenin and c-myc was decreased, but APC was increased obviously when we knockdown WntlOb gene in Ishikawa3-H-12cell line..Conclusion:These results showed that WntlOb gene may play a great role in the genesis and development of EC. This mechanism included the activity of Wnt/β-catenin signal pathway and the regulation to their downstream genes. The over-expression of WntlOb in EC cell lines may activat Wnt/β-catenin signal pathway, inhibit the expression of APC, promoted β-catenin expression and activate the transcription and translation of c-myc gene. Thus,we may draw the conclusion that WntlOb could promote the genesis and development of EC via activation canonical Wnt/β-catenin signal pathway.