Inhibition Of Snail Expression By RNA Interference Repress Epithelial-mesenchymal Transition And Invasion Ability Of Human Gastric Carcinoma Cell

Objective (1)To investigate the expression of Snail and E-cadherin protein in gastriccarcinoma and their relationship with clinical pathological features;(2) to investigate thefrequency of aberrant methylation of E-cadherin in gastric carcinoma;(3) to investigate theeffect of RNA interference (RNAi) targeting transcription factor Snail on epithelial-mesenchymal transition (EMT) and invasion ability of human gastric carcinoma cellSGC-7901 in vitro;(4) to study the establishment of stable high level green fluorescentprotein (GFP)-expressing cell line of gastric carcinoma and pass on its culture continuously.Methods (1)Snail and E-cadherin protein was detected in 152 gastric carcinoma and 30normal gastric tissues by using immunohistochemical method.(2) Aberrant methylationof E-cadherin was examined by methylation specific-PCR (MSP) in gastric carcinoma andnormal gastric tissues (n=54).(3) RNA interference plasmid that can express smallinterfering RNA (siRNA) targeting Snail (Snail siRNA vector) or express siRNA that doesnotmatch any known human coding mRNA (control siRNA vector)wasdesigned,constructed,and lipotransfected into SGC-7901 cell line.SGC-7901-siSnail cellsexpressing Snail suppressed or SGC-7901-si-Control cells expressing Snail uninfluencedwas selected by neomycin resistance.In SGC-7901-nontransfection,SGC-7901-siSnail andSGC-7901-siControl group SGC-7901 cells,Snail,alpha-smooth muscle actin (a-SMA)and E-cadherin expression were examined by reverse transcription-polymerase chainreaction (RT-PCR) and Western blot,invasion ability was examined by Boyden chambermodel.(4) SGC-7901 cell line of gastric carcinoma was transfected by plasma vector with integrated GFP cDNA.GFP-expressing cancer cells were detected by fluorescentmicroscopy through G418 selecting and cloned culture.Results (1) The expression of Snail protein in gastric carcinoma was significantlyhigher than that in normal gastric tissues (p＜0.05);Expression level of Snail wassignificantly related to differential degree,histology type,clinical stage,lymph nodemetastasis,but not correlate with sex,age,tumour size.The expression of E-cadherinprotein in gastric carcinoma was significantly lower than that in normal gastric tissues(p＜0.05);Expression level of E-cadherin was significantly related to differential degree,histology type,lymph node metastasis and but not correlate with sex,age,tumour size,clinical stage.(2) Aberrant methylation of E-cadherin was present in 48.1%(26/54) and11.11%(6/54) in gastric carcinoma and normal gastric tissues respectively.There wassignificant difference between gastric carcinoma and normal gastric tissues group.Therewas significant correlation between E-cadherin methylation frequencies and pathology type,clinical staging,depth of tumour invasion and lymphatic metastasis.No significantassociation was found between E-cadherin methylation frequencies and sex,age.(3)SGC-7901-nontransfection group:Both Snail and a-SMA expression were strong positive,but E-cadherin expression was poor positive.SGC-7901-si Snail group Compared with thatin SGC-7901-nontransfection group,Snail and a - SMA expression,and the numbers ofSGC-7901 cells permeating septum of Boyden chamber were decreased,but E-cadherinexpression increased significantly (p＜0.01).SGC-7901-si Control group:Compared withthat in SGC-7901- nontransfection group,Snail,a-SMA and E-cadherin expression,andNCS were similar.(4) Most of the cancer cells were dead 5 days after being transfected byGFP,scattered or clustered green fluorescence can be seen by microscopy.In culture for 65days,almost all of the clone 2-8 cells expressed high-intensity GFP fluorescence andstability.Conclusions (1)E-cadherin and overexpression of Snail might be impomalignanttransformation and invasion and matastasiscombined detection of E-cadherinherin and Snail has positiinvasion and metastasis in gastric carcinoma.(2) Methylation of E-cadherinmay play a role in the pathogenesis of gastric carcinoma.(3) RNAi targeting Snail caninhibit EMT and invasion of SGC-7901 cells efficiently in vitro.Snail might play a crucialrole in EMT and invasion of lung carcinoma and suppression of Snail expression might bea promising strategy for the treatment of human lung carcinoma.(4)SGC-7901-GFP cellline can provide a basis for establishing an ideal animal model for research of tumorinvasion and metastasis.