FOXD3 Is A Tumor Suppressor Of Colon Cancer By Inhibiting EGFR-Ras-Raf-MEK-ERK Signal Pathway

Objective:To investigate the mechanism of FOXD3 in colon cancer,FOXD3 knockdown cell lines were structured by transfection with FOXD3 siRNA and ShRNA.Methods:（1）Decrease the expression of FOXD3 by small interfering RNA in HCT116 and Caco₂ cells.The expression of FOXD3 was monitored by quantitative real-time polymerase chain reaction（q RT-PCR）and western blot.（2）Cell proliferation was examined by SRB and cloning formation experiment;Cell invasion was examined by transwell analysis.（3）Cell cycle and apoptosis were evaluated by flow cytometry（FACS）.（4）Several proteins which are relation to cell proliferation,apoptosis and metaitasis were detected by western blot.（5）FOXD3 knockdown stable cell lines were injection into the nude mice for the Xenograft experiment.Mice living conditions were observed everyday.Two weeks after injection,mice bearing tumors were sacriced for Western Blot analysis.（6）Collect the patient samples of colon cancer.Then EGFR/Ras/Raf/MEK/ERK pathway were detected by IHC.Results:（1）The Si FOXD3 transfected HCT116 and Caco₂ cells were evaluated by Quantitative Real Time-PCR（q RT-PCR）and Western Blot.The results showed that the expression of FOXD3 in mRNA and protein were significantly inhibited in HCT116 and Caco₂ cells.The difference was statistically significant.（2）The results also showed that silencing FOXD3 can promote cell proliferation（P<0.01,P<0.01）,exhibited faster cell cycle（P<0.05,P<0.05）,induced the cell invasion（P<0.05,P<0.01）,and decreased cell apoptosis（P<0.05,P<0.05）.（3）The result of Western Blot experiment showed that FOXD3 silence could significantly increase protein expression of EGFR and Ras/Raf/MEK/ERK,reduce E-cadherin and cleaved Caspase3 protein expression,increase N-cadherin protein expression.Knocking down EGFR could reduce the activation of K-Ras expression.The difference was statistically significant.（4）Xenograft tumor models were generated.Compared to the control group,tumor growth rate was faster and tumor volume and weight was higher in Sh FOXD3group（P<0.01）.Western Blot results demonstrated that the expression K-Ras,P-ERK,EGFR were reduced in Sh FOXD3 group compared to the control group.（5）Immunohistochemical experiments showed that the expression of FOXD3 was reduced,however,the EGFR/Ras/Raf/MEK/ERK pathway was activated in human cancer tissue.There was statistical difference.Conclusions:（1）FOXD3 could inhibit the proliferation of colon cancer cells by regulating EGFR/Ras/Raf/MEK/ERK signal pathway.（2）FOXD3 played a protective role in colon cancer cells invasion by regulating E-cadherin/N-cadherin signal pathway.