ABSTRACT
Objective TGF-β1 can promote EMT,then strengthen the invasion and metastasis ability of cancer ceils.However,the mechanism for TGF-β1 in gastric cancer still keeps unclear.Aim of this study was to investigate the expression of epithelial to mesenchymal transition (EMT)marker,LMO1 and metastasis related genes on the human gastric cancer cell cell line MKN28 treated with TGF-β1,and test whether down-regulate LMO1 expression can affect the pro-EMT and pro-metastatic roles of TGF-β1 in MKN28 cells.Methods Primary human gastric cancer cell line MKN28 was cultured in vitro.Cells were treated with TGF-β1 to induce cells to undergone EMT.Cells were divided into four groups:control group (5 ％ BSA),TGF-β1 induced group (10 μg/L),negative transfect group (TGF-β1 +negative transfect siRNA),and LMO1-siRNA transfect group (TGF-β1+ LMO1-siRNA).Real time-PCR and Western blot was used to examine the difference of EMT marker (E-cadherin and N-cadherin),LMO1 and metastasis related genes (MMP-9 and VEGF)expression.Transwell assays were performed to identify the differences and changes of invasive and metastatic ability in gastric cancer cell line MKN28.Western blot was used to examine the expression levels of MMP-9 and VEGF.Results TGF-β1 stimulation induced classical EMT morphological change,as was confirmed by E-cadherin decrease and N-cadherin,LMO1,MMP-9,VEGF increase (P＜0.01).Accompanied with the EMT,cell invasion and migration ability was markedly increased (P＜0.01).However,Down-regulation of LMO1 expression reversed the pro-migratory effect of TGF-β1 to a great degree (P＜0.01).Conclusion LMO1 played a central role in coordinating TGF-β1 induced EMT and pro-migratory effects in gastric cancer MKN28 cells.Using siRNA to downregulate the expression of LMO1 can inhibit the invasion and metastasis ability of gastric cancer MKN28 cells.
ABSTRACT
Objective To investigate the expression level of LMO1 in gastric cancer tissues and human gastric cancer cell lines,and explore the invasive and metastatic potential of LMO1 gene silencing by small interfering RNA on the human gastric cancer cell line MKN28.Methods Immunohistochemical technique was applied to detect the expression of LOM1 protein in gastric cancer tissues and tumor adjacent tissues of paraffin specimens in 30 cases.The expression levels of LMO1 in human gastric cancer cell lines AGS,BGC-823,SGC-7901,MKN28 and human gastric mucosal epithelial cells GES were detected by realtime-PCR and Western blot.Using LipofectamineTM 2000,LMO1 siRNA was transfected into MKN28 cellsin vitro (siRNA transfect group).Negative control was established.Real time-PCR and Western blot was used to examine the difference of LMO1 expression.Transwell assays were performed to identify the differences and changes of invasive and metastatic ability in gastric cancer cell line MKN28.Western blot was used to examine the expression levels of E-cadherin,MMP-9 and VEGF.Results Positive rate of LOM1 protein in gastric cancer tissues(77％)was higher than that in tumor adjacent tissues (17％) (x2 =21.70,P ＜ 0.01).Positive rate of Vavl protein was higher in lymphatic metastasis group than in non-lymphatic metastasis group(x2 =5.83,P =0.02).Compared with GES,the expression level of LMO1 increased significantly in gastric cancer cell lines,especially in MKN28 (P ＜ 0.01).The expression levels of LMO1 mRNA and protein in LMO1-siRNA transfected MKN28 cells were lower than the matched negative control cells (P ＜0.01).The invasive and metastatic potentials of LMO1-siRNA transfected MKN28 cellssignificantly decreased (t =-11.53,P ＜0.01;t =-10.68,P ＜0.01).The expression levels of E-cadherin were higher than the matched negative control cells;and MMP-9,VEGF protein in LMO1-siRNA transfected MKN28 cells were lower than the matched negative control cells (P ＜ 0.01).Conclusions LMO1 has higher expression level in gastric cancer tissues and some gastric cancer cell lines,and down-regulation of LMO1 can inhibit the invasion and metastasis ability of gastric cancer.