| Objective:First of all,To investigate the role and mechanism of Wnt5 a in the development and progression of endometrioid adenocarcinoma,then to explore the role of Wnt5 a in regulating invasion and metastasis of Ishikawa cell line.Methods:1.The normal endometrium,atypical hyperplasia endometrium,and endometrioid adenocarcinoma wax blocks were collected in groups,and the related clinical and pathological features were collected and summarized;2.Immunohistochemistry was used to detect the expression of Wnt5 a,EZH2,?-catenin,and E-cadherin in each group of wax samples.The relationships of Wnt5 a and clinicopathological factors and EMT-related proteins EZH2,?-catenin,and E-cadherin were also statistically analyzed;3.The Ishikawa endometrial carcinoma cell line was divided into negative control group,empty vector group and Wnt5 a plasmid group.The empty vector and Wnt5 a plasmid were transfected into the latter two groups,and the last group had Wnt5 a was expressed and the transfection efficiency was detected by flow cytometry;4.After transfection was observed by fluorescence microscope,the transfection was stopped at the right time and the transfection efficiency was detected by flow cytometry;5.Western Blot was used to detect the expression differences of Wnt5 a,EZH2,?-catenin and E-cadherin proteins in endometrial cancer cells of each group.The effect of overexpression of Wnt5 a on EMT-related proteins and Wnt/?-catenin pathway was analyzed;6.The effect of overexpression of Wnt5 a on the value-added ability of Ishikawa cell line was detected by CCK8 value-added assay;7.Transwell chamber migration assay and invasion assay were used to detect the effect of overexpression of Wnt5 a on migration and invasion of Ishikawa cell line;8.The effect of Wnt5 a overexpression on EMT-related proteins and Wnt/?-catenin pathways in endometrial cancer cell lines was tested by Real-time PCR at the gene level.Results:1.The expression of Wnt5 a protein in endometrial cancer group was lower than normal endometrium group and atypical hyperplasia endometrium group(P<0.01).There was no significant difference between the latter two groups.The E-cadherin protein expression trend was the same,but the EZH2 and ?-catenin protein expression trends were opposite.2.The expression of Wnt5 a protein was not related to age and depth of myometrial invasion,but the positive expression rate of Wnt5 a was gradually decreased in cases with well-differentiated,moderately-differentiated and poorly differentiated(P<0.05),and the positive expression was the same in each stage of the pathological stage(P<0.05).And its positive rate in non-metastatic cases was significantly higher than that in metastatic cases,the difference was statistically significant.3.Western blot results showed that the gray ratios of Wnt5 a and E-cadherin proteins in the transfection group were higher than those in the negative control group and the empty vector group(P<0.01),and there was no significant difference between the latter two groups;but the gray ratios of EZH2 and ?-catenin proteins in the transfection group was lower than those in the negative control group and empty vector group(P<0.01),and there was no significant difference between the latter two groups.4.Transwell chamber migration assays and invasion experiments showed that the numbers of cells in the overexpression group were significantly lower than those in the negative control group and the empty vector group(P<0.01).There was no significant difference between the latter two groups.Real-time PCR results showed that the RQ values of Wnt5 a and E-cadherin mRNA inthe transfection group were significantly higher than those in the negative control group and the empty vector group(P<0.01),and there was no significant difference between the latter two groups;while the RQ values of EZH2 and ?-catenin in the transfection group was significantly lower than those in the negative control group and the empty vector group(P<0.01),and there was no significant difference between the the latter two groups.Conclusions:1.Wnt5 a is generally lowly expressed in endometrial cancer.As a tumor suppressor gene,its negative expression may be related to tumorigenesis and promote tumor progression.Wnt5 a may provide an effective and new indicator for the diagnosis and treatment of endometrial cancer.2.The results of western Blot and Real-time PCR showed that overexpression of Wnt5 a in endometrial cancer cell line Ishikawa can antagonize the Wnt/?-catenin signaling pathway,down-regulate the expression of EMT-related transcription factors EZH2 and ?-catenin,and up-regulate E-cadherin expression,then inhibited the EMT process.3.Overexpression of Wnt5 a inhibited proliferation,migration and invasion of endometrial cancer cells. |
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