| Objective: Gastric cancer is one of the most common malignancies in digestive system neoplasms.Its morbidity and mortality have been high for many years.The mortality rate of gastric cancer causes the second leading of cancer mortality in China.Because early gastric cancer rarely have obvious symptoms,more than half of patients with gastric cancer are found in advanced cancer,or even lose the surgical opportunities,while treatment are more rely on chemotherapy.Cisplatin is the first-line drugs for the treatment of gastric cancer,but the efficiency is still not more than 20%.Gastric cancer resistance to cisplatin is an important reason for the failure of gastric cancer chemotherapy.With the study of the mechanism of cisplatin resistance of gastric cancer,a series of drug resistance-related genes have been found.Among them,Annexin A family plays an important role in cisplatin resistance of gastric cancer.However,the relationship of ANXA11 and drug resistance of human gastric cancer cell line AGS to cisplatin has not yet been reported.Therefore,this study chosed human gastric cancer cell line AGS as the object and detected drug susceptibility of human gastric cancer cell line AGS to cisplatin after the expression of ANXA11 gene inhibited by siRNA interference technology.After inhibiting the expression of ANXA11 gene,we detected the drug sensitivity of each group of human gastric cancer cells AGS to cisplatin.Compared with negative control group and blank control group,drug sensitivity of transfection group cells AGS to cisplatin was significantly increased and we preliminary study its related mechanism.The study provided some experimental basis for the remission of drug resistance on gastric cancer cell AGS to cisplatin.Methods: Human gastric cancer cell line AGS and human gastric cancer cell line MGC-803 were in cultured.The expressions of ANXA11 mRNA and protein of two cell lines were detected by qPCR and Western blot.Design and synthese of ANXA11-specific interference siRNA sequences and its negative control sequence,human gastric cancer cell AGS was transiently transfected with ANXA11 siRNA and its negative control by means of Lipofectamine2000.Western and qPCR were used to detect the expression of ANXA11 protein and mRNA after transfection 24,48,72 hours and detect the interference effects.The experiment groups were as follows: transfection group(human gastric cancer cell AGS transfected by ANXA11-siRNA-2),Negative control group(human gastric cancer cell AGS transfected by ANXA11-siRNA-NC),Blank control group(human gastric cancer cell AGS).After transfection 48 hours,CCK-8 method was used to detect the inhibitory rate of each group cells under different concentrations of cisplatin,and then calculate the IC50 and IC20(considering the damage of IC50 cisplatin for cell is bigger,subsequent experiments with IC20 processing cells)of each group cells.The experiment groups were as follows: combined group(human gastric cancer cell AGS transfected by ANXA11-siRNA-2 and disposed with IC20cisplatin),transfection group(human gastric cancer cell AGS transfected by ANXA11-siRNA-2),negative control group(human gastric cancer cell AGS transfected by ANXA11-siRNA-NC)and blank control group(human gastric cancer cell AGS).Flow cytometry was used to detect the apoptosis rate of each group of cells under the IC20 cisplatin.The total mRNA and protein of each group were extracted after transfection 48 hours,and the expression of Bax and Bcl-2 mRNA and protein were detected respectively by qPCR and Western.Result:1 ANXA11 mRNA relative expression in human gastric cancer cell AGS was0.086 + 0.022,while in human gastric cancer cell MGS-803 was 0.223 +0.008.ANXA11 protein relative expression in human gastric cancer cell AGS was 1.969 + 0.105,while in human gastric cancer cell MGS-803 was 0.935 +0.078.Compared with the human gastric cancer cell line MGS-803,the expression of ANXA11 mRNA and protein in human gastric cancer cell line AGS were significantly higher(P<0.01).2 ANXA11-siRNA screening2.1 ANXA11-siRNA-1,ANXA11-siRNA-2,ANXA11-siRNA-3 and ANXA11-siRNA-123 were transfected into gastric cancer cell line AGS for24,48,72 hours,and the expression of ANXA11 mRNA was tested by qPCR.The results showed that,compared with the negative control group and blank control group,the expression of ANXA11 mRNA in each group was inhibited in different degress by ANXA11 siRNA(F=20.891,P=0.000).Among them,ANXA11 siRNA transfection 48 hours is the best and inhibition rate of ANXA11-siRNA-2 was the highest,reaching 82.36%.2.2 ANXA11-siRNA-2 in different concentration(80nM,100 n M,120 n M,and 150 n M)was transfected into human gastric cancer cell AGS for 24,48,72 hours,and the expression of ANXA11 protein was tested by Western blot.The results showed that,compared with the negative control group and blank control group,each group has inhibitory effect to the expression of ANXA11 protein in different degress(F=51.641,P=0.000).Among them,ANXA11-siRNA-2 transfection 48 hours is the best and the inhibition efficiency of100 n M ANXA11-siRNA-2 was the highest,reaching 57.03%.3 ANXA11-siRNA-2 was transfected into human gastric cancer cell AGS for24,48,72 hours,the IC50 cisplatin to each group cells as follow: in transfection group was 1.923±0.205μg/ml,in negative control group was3.817±0.413μg/ml,in blank control group was 4.280±0.216μg/ml.The IC20 cisplatin to esch group cells as follow: in transfection group was0.105±0.012μg/ml,in negative control group was 0.289±0.154μg/ml,in blank control group was 0.233±0.329μg/ml.The sensitivity of human gastric cancer cell AGS to cisplatin after ANXA11 siRNA transfection 48 hours was significantly higher than negative control group and blank control group(F=149,P=0.000).4 The apoptosis rate of transfection group was(16.557±0.979)%,the apoptosis rate of combined group was(40.416±1.652)%,while that of negative control group was(6.560±0.680)% and that of the blank control group was(5.387±0.620)%.Compared with the negative control group and blank control group,the apoptosis rates of combinated group and transfection group were significantly increased(F=698.733,P=0.000).Compared with the single transfection group,the apoptosis rate of combinated group was significantly increased(P=0.0 00).5 Effect of ANXA11-siRNA-2 transfection on the expression of Bax and Bcl-2 gene qPCR detected the expression changes of Bax,Bcl-2 mRNA after ANXA11-siRNA-2 inhibited ANXA11 expression.The results show that,compared with the negative control group and blank control group,the expression of combination group and transfection group Bcl-2 mRNA was significantly decreased after 100 n M ANXA11-siRNA-2 transfected into AGS cells for 48 hours(F=41.046,P=0.000),while the expression of Bax mRNA was significantly increased(F=34.205,P=0.000).Compared with transfection group,the expression of combinated group Bcl-2 mRNA was significantly lower(P=0.003),while the expression of Bax mRNA was significantly increased(P=0.014).Western blot detected the expression changes of Bax,Bcl-2 protein after ANXA11-siRNA-2 inhibited ANXA11 expression.The results show that,compared with the negative control group and blank control group,the expression of combinated group and transfected group Bcl-2 protein was significantly decreased after 100 n M ANXA11-siRNA-2 transfected into AGS cells for 48 hours(F=30.184,P=0.000),while the expression of Bax protein was significantly increased(F=41.424,P=0.000).Compared with transfection group,the expression of combination group Bcl-2 protein was significantly lower(P=0.002),while the expression of Bax protein was significantly increased(P=0.049).Conclusion:1 Comepared with human gastric cancer cell line MGC-803,the espression of ANXA11 mRNA and protein in human gastric cancer cell line AGS are more higher.Inhibition of ANXA11 gene expression can improve the apoptosis rate of gastric cancer AGS cells.2 By inhibiting the expression of ANXA11 gene,we can enhance the inhibitory effect of cisplatin on gastric cancer cell line AGS,so as to improve the sensitivity of human gastric cancer cell line AGS to cisplatin.ANXA11 may be a new gene involved in human gastric cancer cell AGS cisplatin resistance.3 ANXA11 gene induced the drug resistance of human gastric cancer AGS cells line to cisplatin may relate to increasing antiapoptotic Bcl-2 gene expression and inhibiting apoptotic Bax gene expression. |
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