| Objective: We investigate the relationship between the expression of DENND1 A in endometrial carcinoma(EC)tissues and its clinicopathological characteristics and prognosis by immunohistochemistry using the endometrial tissue specimen library of Tianjin medical university general hospital.The effects of DENND1 A on the proliferation,migration and invasion of EC cells were investigated by lentivirus transfection in vitro.The effects of DENND1 A on the expression and activation of PI3K/Akt signaling pathway related proteins were detected by Western blot,which provided a new idea for the occurrence and development of endometrial cancer.Methods: 1.Relevant case data of EC patients 228 cases and matched normal controls 148 cases and patients with atypical endometrial hyperplasia 46 cases were collected;Immunohistochemical methods were used to detect the expression of DENND1 A in these three tissues,to analyze the correlation between the expression of DENND1 A in EC tissues and the clinicopathological characteristics of EC patients,and to explore the relationship between DENND1 A and the survival and prognosis of EC patients.2.Western blot method was used to detect the expression of DENND1 A in EC cell lines AN3 CA,ECC-1,Ishikawa,HEC-l A,HEC-l B,KLE.Endometrial carcinoma HEC-l B and HEC-l A cell lines were selected for up-regulation and down-regulation of DENND1 A expression by lentivirus transfection.The effects of up-regulation and down-regulation were detected by q RT-PCR and Western blot.Clonal formation assay was used to detect the proliferation of EC cells after up-regulation and down-regulation of DENND1 A.Transwell chamber was used to detect the migration and invasion of EC cells after up-regulation and down-regulation of DENND1 A.Western Blot was used to detect the expression of t-Akt,p-Akt and Cyclin D1 signaling pathway proteins in endometrial cancer cells before and after up-regulation of DENND1 A.Results:1.The expression level of DENND1 A in EC tissue was higher than that in normal control and atypical hyperplasia endometrium,but there was no statistical difference(P > 0.05).In EC tissues,the positive expression level of DENND1 A with FIGO I-II phase was significantly higher than that with FIGO III-IV phase,and the difference was statistically significant(P < 0.05).In type I EC tissues,the positive expression level of DENND1 A in FIGO I-II patients was significantly higher than that in FIGO III-IV patients,and the positive expression level of DENND1 A in patients without lymph node metastasis was significantly higher than that in patients with lymph node metastasis(P<0.05).In type I EC tissues,the overall survival rate in the DENND1 A positive expression group was significantly higher than that in the negative expression group,and the difference was statistically significant(P < 0.05).2.Among the six EC cell lines,the expression level of DENND1 A in HEC-1A cells was the highest,and the expression level of DENND1 A in HEC-l B cells was the lowest.After lentivirus transfection,DENND1 A expression was increased in HEC-l B cells and decreased in HEC-1A cells.Compared with the negative control group,the number and volume of HEC-l B cells in endometrial carcinoma cells were decreased when the expression of DENND1 A was up-regulated,and the difference was statistically significant(P<0.05).Compared with the negative control group,Transwell chamber experiments showed that the transmembrane number of HEC-l B cells was significantly reduced(migration: 65.9±3.7162 vs.33.3±5.9080;Invasion: 25.3±3.4503 vs.9.0±2.0000),the difference was statistically significant(P<0.05).When DENND1 A expression was down-regulated,the number and volume of HEC-1A cell clone formation increased compared with the negative control group,and the difference was statistically significant(P< 0.05).Compared with the negative control group,Transwell chamber experiments showed that the transmembrane number of HEC-1A cells increased(migration: 13.6±3.2094 vs.32.4±3.9115;Invasion: 6.3±1.0328 vs.17.5±5.7183),the difference was statistically significant(P< 0.05).After the up-regulation of DENND1 A,compared with the negative control group,the expression level of DENND1 A increased,while the expression levels of p-Akt and Cyclin D1 decreased,the difference was statistically significant(P < 0.05),and there was no significant change in the expression level of t-Akt(P > 0.05).Conclusion: 1.DENND1 A may be associated with the occurrence and development of EC.Positive expression of DENND1 A is associated with early stage,no lymph node metastasis in EC patients.The cumulative survival rate is higher than negative expression of DENND1 A,especially in type I EC,suggesting that DENND1 A may be an early event in type I EC.DENND1 A may be a molecular marker for the prognosis of type I EC.2.After the up-regulation of DENND1 A in HEC-l B cells,the ability of cell clone was decreased,and Transwell cell transmembrane number was decreased,which indicates that DENND1 A could inhibit the migration and invasion of endometrial cancer cells.After down-regulation of DENND1 A in HEC-1A cells,the clonal formation assay showed that the proliferation capacity of EC cells increased,and Transwell assay showed that the transmembrane number of EC cells increased,which indicates that DENND1 A could inhibit the migration and invasion of endometrial cancer cells.Up-regulation of DENND1 A inhibited Akt activation and Cyclin D1 expression in endometrial cancer cells,suggesting that DENND1 A may inhibit the progression of endometrial cancer cells through the PI3K/Akt signaling pathway. |
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