| Objective(s): At the tissue level,the expression difference of Twist1 in gallbladder cancer and adjacent tissues was clarified,and the correlation between Twist1 and clinicopathological factors in gallbladder cancer patients was analyzed.Secondly,Twist1 shRNA lentivirus was constructed at the cell level in vitro to investigate the effects of targeted silencing Twist1 on the biological behavior of gallbladder cancer cells,and to further investigate the effects of targeted silencing Twist1 on the expression levels of Bmi1,HIF-1α,WWOX,E-Cadherine and NCadherine.Methods: Immunohistochemistry was used to evaluate whether the expression of Twist1 protein was different in gallbladder carcinoma tissues and adjacent tissues,and to analyze the relationship between Twist1 expression and clinicopathological features in gallbladder carcinoma tissues.Twist1 shRNA lentivirus was constructed in vitro.The Twist1 shRNA lentivirus vector infected BGC-SD cells as the experimental group(Twist1 group),the no-load lentivirus-infected BGC-SD cells as the negative control group(NC group),and the BGC-SD cells without any treatment as the blank control group(Mock group).The mRNA and protein expression levels of Twist1 in each group were detected by qRT-PCR and Western blot,and the efficiency of lentivirus infection was detected.Cell scarring and Transwell migration and invasion assay were performed to detect the migration and invasion ability of cells.mRNA and protein expression levels of Bmi1,HIF-1α,WWOX,E-Cadherine and N-Cadherine were detected by qRT-PCR and Western blot.Results: 1.Twist1 expression in gallbladder carcinoma was significantly higher than that in paracancer tissue.2.The expression of Twist1 protein in gallbladder carcinoma is related to the T stage,lymph node metastasis and TNM stage.3.After lentivirus infection with BGC-SD cells,qRT-PCR was used to detect Twist1 mRNA expression in the three groups.The results indicated that the relative expression level of Twist1 mRNA in the Twist1 group was 0.35±0.02.NC group: 1.26±0.23;Mock group: 1.00±0.48.The mRNA relative expression level of Twist1 in the experimental group was significantly lower than that in the control group,and the difference was statistically significant(P≤0.01).Western blot was used to detect the expression of Twist1 protein in the three groups.The results indicated that the relative expression level of Twist1 protein in the Twist1 group was 0.35±0.20.NC group: 0.95±0.21;Mock group: 1.01±0.18.The protein relative expression level of Twist1 in the experimental group was significantly lower than that of the two control groups,with statistical significance(P≤0.0001).The results showed that Twist1 shRNA lentivirus successfully infected BGC-SD cells and inhibited Twist1 expression at RNA and protein levels.4.Cell scratch experiment results: the scratch healing rate was calculated by the scratch area.Compared with the negative control group and the blank control group,the scratch healing rate of the experimental group was significantly smaller,and the difference was statistically significant(P ≤0.0001).Results of Transwell cell migration experiment: Compared with negative control group and blank control group,the number of BGC-SD cell migration in experimental group was significantly smaller,and the difference was statistically significant(P ≤ 0.0001).Results of Transwell cell invasion experiment: Compared with negative control group and blank control group,the number of cell invasion in experimental group was significantly lower,the difference was statistically significant(P≤0.0001).The results showed that targeted silencing Twist1 could significantly inhibit the migration and invasion of gallbladder carcinoma cells.5.qRT-PCR was used to detect the mRNA expression of Bmi1,HIF-1α,WWOX,E-Cadherine and NCadherine in the three groups of cells.The results indicated that: The mRNA relative expression levels of Bmi1,HIF-1α and N-Cadherine in the experimental group were significantly lower than those in the two control groups,with statistical significance(P≤0.01).The mRNA relative expression levels of WWOX and E-Cadherine in the experimental group were significantly higher than those in the control group,with statistical significance(P≤0.01).The results showed that targeted Twist1 knockdown inhibited the mRNA expression of Bmi1,HIF-1α and N-Cadherine,but promoted the mRNA expression of WWOX and E-Cadherine.6.Western blot was used to detect the expression of Bmi1,HIF-1α,WWOX,E-Cadherine and N-Cadherine in the three groups of cells.The results indicated that: The protein relative expressions of Bmi1,HIF-1α and N-Cadherine in the experimental group were significantly lower than those in the two control groups,and the difference was statistically significant(P≤0.01).The protein relative expression levels of WWOX and E-Cadherine in the experimental group were significantly higher than those in the control group,with statistical significance(P≤0.01).The results showed that targeting Twist1 inhibited the protein expression of Bmi1,HIF-1α and N-Cadherine,but promoted the protein expression of WWOX and E-Cadherine.Conclusion(s): 1.Twist1 protein was highly expressed in gallbladder carcinoma tissues,and its expression was correlated with T stage,lymph node metastasis and TNM stage.2.Targeted silencing of Twist1 can inhibit migration and invasion of BGC-SD cells.3.Targeted silence of Twist1 can inhibit the expression of Bmi1 and HIF-1α,and promote the expression of WWOX.4.Targeting Twist1 to suppress the expression of mesenchymal marker N-Cadherine during EMT,and promote the expression of epithelial marker E-Cadherine.5.Targeting Twist1 silencing to inhibit the invasion and migration of BGC-SD cells may be achieved by regulating the Bmi1/WWOX pathway to inhibit EMT process. |
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