The Study Of Mechanism And Effects Of Twist Gene On Invasion And Metastasis Of Gastric Carcinoma Cells

Introduction Gastric carcinoma(GC)is a frequent neoplasm that severely threatens people's health,and its mechanism is still unknown. The Twist protein is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix proteins,and it plays an important role in the embryogenesis.Twist is regarded as an oncogene protein, regulating the apoptosis of tumor cells.It has been shown that Twist is a key regulator of epithelial-mesenchymal transition and responsible for the invasion and metastasis of tumor.Despite some research of Twist,we know little of whether Twist contributes to the invasion and metastasis of gastric carcinoma.Objective Our investigation attempts to study the effect of transfected Twist gene on invasion and metastasis of human gastric carcinoma cells and the role of Twist gene in gastric carcinogenesis.Methods After bacteria transformation,use restriction enzyme technique,gel electrophoresis technique and gene sequencing identification the Twist sense plasmid;The SGC7901 cells were transfected with PcDNA3-Twist sense plasmid by lipofectamine technique,and gained the stable TwistS-SGC7901 cell model with high expression of Twist protein;The mRNA of Twist were detected by RT-PCR;The expression of Twist protein were detected by immunofluorescence and Western blotting;The expression of EMT marker(Ecadherin,α-SMA)were detected by RT-PCR;Adherent ability of transfected cells were detected by MTT method;The ability of migration and invasion of transfected cells was examined by Bodern method;Nude mice metastasis models were established by abdominal cavity transfer method;The expression ofβ-catenin protein were detected by immunofluorescence;The Tcf-4 DNA binding ability was detected by EMSA;The expression of Tcf-4 downstream target gene cyclinD1 was detected by RT-PCR;The expression of Tcf-4 downstream target gene vimentin and mmp-2 was detected by Western blotting;the expression of VEGF was analysised with ELISA.After bacteria transformation,extracted the Twist antisense plasmid; The MKN45 cells were transfected with PcDNA3-Twist antisense plasmid by lipofectamine technique,and gained the stable TwistAS-MKN45 cell model with low expression of Twist protein;The mRNA of Twist were detected by RT-PCR;The expression of Twist protein were detected by Westem blotting;The Tcf-4 DNA binding ability was detected by EMSA;The expression of Tcf-4 downstream target gene cyclinD1 was detected by RT-PCR;The expression of Tcf-4 downstream target gene vimentin and mmp-2 was detected by Western blotting;the expression of VEGF was analysised with ELISA;The expression of E-cadherin protein were detected by Western blottingWe analyzed the expressions of Twist and Ecadherin proteins and their correlation with clinical features on formalin-fixed,paraffin-embed-ed tissue sections from 76 patients with Gastric cancer using immunohistochemistry method.Results The cell models(TwistS-SGC7901)steadily expressing high Twist protein were obtained;Compared with SGC7901 and PcDNA3-SGC7901 cells,the cell adherence,migration,invasion and metastasis ability of TwistS-SGC7901 cells were increased obviously (P＜0.05);Twist played an important role in promoting SGC7901 cells epithelial-mesenchymal transition;SGC7901 cells transfected with Twist sense plasmid increased Tcf-4 transcription factor' DNA binding ability, and also promoted the expression of Tcf-4 downstream target genes cyclinD1,VEGF,Vimentin and mmp-2.The cell models(TwistAS-MKN45)steadily expressing low Twist protein were obtained;MKN45 cells transfected with Twist antisense plasmid inhibited Tcf-4 transcription factor' DNA binding ability,and also inhibited the expression of Tcf-4 downstream target genes cyclinD1, VEGF,Vimentin and mmp-2.Twist protein positive signal was localized in cytoplasm and in nucleus.40 out of 76(60.52%)gastric cancer tissues expressed Twist protein.But no signals were detected in gastric mucosa.The expression level of Twist protein was related to tumor differention degree,invasion extention,lymph node metastasis;but no significant correlation was observed between Twist and age or gender.30 of 76(47.37%)gastric cancer tissues expressed Ecadherin protein,but all the gastric mucosa expressed Ecadherin protein.The expression level of Ecadherin protein was related to tumor differention degree,invasion extention and lymph node metastasis.But no significant correlation was observed between Ecadherin and age or gender.However,the Twist level was negative related to the Ecadherin level.Conclusion1 Overexpression of Twist could promote the cell adherence, migration,invasion and metastasis ability of SGC7901 cells.2 Twist could induce EMT of SGC7901 cells.3 Twist promoted the invasion and metastasis of SGC7901 cells probably through the regulation of Wnt signaling dependent mechanism.4 The expression of Twist and E-cdhefin in gastric cancer was related to tumor differention degree,invasion extention and lymph node metastasis.Twist could promoted the invasion and metastasis of gastric cancer by inhibiting the expression of Ecadherin protein.