| Research basis:1.In the early stage,the research group divided the highly differentiated human gastric cancer MKN28 cell lines into experimental group(KAI1-si RNA group)transfected with KAI1 gene lentivirus RNA interference vector plkd-cmv-egfp-2a-puro-u6-shrna(KAI1),negative control group transfected with empty virus vector plkd-cmv-egfp-puro-u6-shrna(MKN28-NC group)and blank control group without special treatment(MKN28-Ctrl group),After 72 hours of virus infection,the expression of green fluorescent protein EGFP in cells of each group was observed under fluorescence microscope,and the stably transfected cell lines were established by dosing screening method.2.In the early stage of the research group,36 female nude mice aged 4-6 weeks were randomly divided into 6 groups with 6 in each group.The highly differentiated human gastric cancer MKN28 cell lines in each group were inoculated into the abdominal cavity of nude mice,with a cell volume of 1×10~7/ml,the growth and metastasis of abdominal transplanted tumor in nude mice were detected by small animal color ultrasonic detector,and the transplanted tumor model in nude mice was successfully constructed.Objective:To investigate the effect of KAI1 tumor suppressor gene on Epithelial-Mesenchymal Transition of human gastric cancer MKN28 cells in vitro and in vivo,and the possible mechanism of KAI1 gene effect on EMT.Methods:1.q PCR was used to detect the transcription levels m RNA of KAI1,key molecular of Wnt/β-catenin signaling pathway(β-catenin)and EMT-related molecules(E-cadherin,N-cadherin)in human gastric cancer MKN28 cells in vivo and in vitro.2.Western blot was used to detect the protein expressions of KAI1,key molecular of Wnt/β-catenin signaling pathway(β-catenin)and EMT-related molecules(E-cadherin,N-cadherin)in human gastric cancer MKN28 cells in vivo and in vitro.3.IHC was used to detect the expression ofβ-catenin,E-cadherin and N-cadherin proteins in the transplanted tumor tissues,and their MOD values were measured.Results:1.The results of q PCR showed that the relative expression of KAI1 m RNA in KAI1-si RNA group was lower than those in MKN28-NC and MKN28-Ctrl groups(P<0.05);the relative expression of EMT epithelial marker E-cadherin m RNA in KAI1-si RNA group was lower than those in MKN28-NC and MKN28-Ctrl groups(P<0.05),while the relative expression of mesenchymal marker N-cadherin m RNA in KAI1-si RNA group was higher than those in MKN28-NC and MKN28-Ctrl groups(P<0.05);the relative expression of key molecules of Wnt/β-catenin signaling pathway(β-catenin)m RNA in KAI1-si RNA group was higher than those in MKN28-NC and MKN28-Ctrl group(P<0.05),there was no statistical significance between NC group and Ctrl group(P>0.05).2.The results of Western blot showed that the relative expression of KAI1 protein in KAI1-si RNA group was lower than that in MKN28-NC group and MKN28-Ctrl group(P<0.05);The relative expression of EMT epithelial marker E-cadherin protein in MKN28-si RNA group was lower than that in MKN28-NC group and MKN28-Ctrl group(P<0.05),while the relative expression of mesenchymal marker N-cadherin protein in KAI1-si RNA group was higher than those in MKN28-NC group and MKN28-Ctrl group(P<0.05).The relative expression of key molecules of Wnt/β-catenin signaling pathway(β-catenin)protein in KAI1-si RNA group was higher than that in MKN28-NC group and MKN28-Ctrl group(P<0.05),There was no statistical significance between NC group and Ctrl group(P>0.05).3.The results of IHC showed that the MOD value of E-cadherin protein in KAI1-si RNA group was lower than those of MKN28-NC group and MKN28-Ctrl group in transplanted tumor tissues(P<0.05);The MOD values ofβ-catenin and N-cadherin protein in KAI1-si RNA group were higher than those in MKN28-NC group and MKN28-Ctrl group(P<0.05),there was no statistical significance between NC group and Ctrl group(P>0.05).Conclusion:1.KAI1 was positively correlated with E-cadherin expression in transplanted tumor tissue and gastric cancer cell line,KAI1 was negatively correlated with N-cadherin expression in transplanted tumor tissue and gastric cancer cell line;Silencing of KAI1 gene expression may promotes Epithelial-Mesenchymal Transition process in gastric cancer cells;2.KAI1 was negatively correlated withβ-catenin expression in transplanted tumor tissue and gastric cancer cell line;KAI1 gene is involved in Epithelial-Mesenchymal Transition process of gastric cancer cells,which may be negatively regulated by Wnt/β-catenin signaling pathway. |
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