DIAPH3 Promotes Proliferation,Migration And Invasion Of Esophageal Squamous Cell Carcinoma And Its Molecular Mechanisms

BackgroundEsophageal cancer（EC）is one of the common gastrointestinal malignancies.its histology is divided into esophageal squamous cell carcinoma（ESCC）and esophageal adenocarcinoma（EAC）,of which ESCC accounts for more than 90%.Diaphanous related formin 3（DIAPH3）is one member of the Diaphanous-related formin（DRF/DIAPH）family,localized to the long arm of chromosome 13（13q21.2）.It has been reported that DIAPH3 plays an important role in many tumors,while the role and molecular mechanisms of DIAPH3 in ESCC are still unknown.This study will be investigated the role of DIAPH3 in the proliferation,migration,and invasion in ESCC.ObjectiveThe expression of DIAPH3 in ESCC tissues will be explored.The ECA109,ECA9706,KYSE150,and KYSE180 ESCC cell lines were used to explore the expression of DIAPH3 in the ESCC tissues,which were also used to explore the effect of DIAPH3 on its proliferation,migration,and invasion capacity.Methods1.The expression of DIAPH3 m RNA in ESCC was predicted by the gene expression profiling interactive analysis（GEPIA）and the cancer genome atlas（TCGA）databases.2.The expression of DIAPH3 protein was analyzed in ESCC by immunoh-istochemical（IHC）.The correlation of DIAPH3 expression and related clinicop-athological features was analyzed.The expression of DIAPH3 protein was dete-cted in 8 pairs of fresh ESCC tissues and paired adjacent tissues by Western blot experiment.3.The expressions of DIAPH3 m RNA and protein were measured by q PCR and Western blot.DIAPH3 interference and overexpression cell lines were constructed.their efficiency was verified.4.Gene set enrichment analysis（GSEA）was performed by Linked Omics database.The effects of overexpression / interference with DIAPH3 on the proliferation,migration and invasion capacity in ESCC cells were examined using CCK-8,flow cytometry,the wounding assay and Transwell invasion assay.5.The expressions of EMT-related proteins and cyclin D1 protein were determined by Western blot experiments.Results1.The results of GEPIA and TCGA databases showed that the expression of DIAPH3 m RNA in ESCC was significantly higher than that in adjacent tissues（P<0.05）.2.The result of IHC showed that DIAPH3 was mainly localized in the cytoplasm,with strong positive staining and yellow.The positive rate of DIAPH3 in ESCC tissue was significantly higher than the adjacent carcinoma tissue（P<0.05）.Western blot showed that the expression of DIAPH3 protein in ESCC was significantly higher than that in paired adjacent tissues（P< 0.05）.3.The results of q PCR and Western blot showed that the expression of DIAPH3 m RNA and protein in ESCC cells from high to low was k YSE150,KYSE180,ECA9706 and ECA109.DIAPH3 interference and overexpression cell lines were successfully constructed（P<0.05）.4.CCK-8 experiment showed that the proliferation speed of ECCC cells in siDIAPH3 was significantly slower than that in NC group.the proliferation speed of ESCC in DIAPH3 group was significantly faster than that in Control group（P< 0.05）.5.The result of GSEA in Linked Omics database showed that DIAPH3 was related to cell cycle（P<0.05）.The result of Western blot showed that the expression of cyclin D1 protein of ESCC cells in si-DIAPH3 group was significantly higer than that in NC group.the expression of cyclin D1 protein of ESCC cells in DIAPH3 group was significantly higher than that in Control group（P<0.05）.The result of flow cytometry showed that the apoptosis rate of ESCC cells in si-DIAPH3 group was significantly higher than that in NC group.The apoptosis rate of ESCC cells in the DIAPH3 group was significantly lower than that in Control group（P< 0.05）.6.The results of the wounding assay and Transwell invasion assay showed that the wounding percent and the numbers of ESCC cells passing through the chamber in the si-DIAPH3 group were significantly lower than in the NC group（P<0.05）.the wounding percent and the numbers of ESCC cells passing through the chamber were significantly higher than in the Control group（P<0.05）.7.The result of Western blot showed that the expression of E-cadherin in siDIAPH3 group was significantly higher than that in NC group.the expression of Vimentin was significantly lower than that in NC group in ESCC cells（P < 0.05）.the expression of E-cadherin in the DIAPH3 group was significantly lower than that in Control group.the expression of Vimentin was significantly higher than that in Control group in ESCC cells（P < 0.05）.Conclusions1.The expression of DIAPH3 in ESCC tissues increased significantly,which was closely related to the degree of tumor differentiation and lymph node metastasis,but not related to tumor size,age and gender.2.DIAPH3 promotes the proliferation of ESCC cells by enhancing the expression of cyclin D1.DIAPH3 promotes the migration and invasion of ESCC cells by EMT process.