| Objective: In recent years,although the incidence of gastric cancer worldwide decreased,the incidence of gastric cancer in China has increased year by year.The incidence and mortality of gastric cancer are second,with lung cancer being the most common cancer and the leading cause of death.Comprehensive treatment based on surgery is the main treatment in gastric cancer.However the diagnosis rate of early gastric cancer in China is less than10%,most patients often lost the chance for radical surgery,which makes chemotherapy plays an important role in the treatment of gastric cancer.Although more than half a century has past since the advent of 5-fluorouracil(5-Fu),it is still an important component of current chemotherapy regimen in gastric cancer.Due to the primary and secondary drug resistance of tumor cells,the effective rate of single agent chemotherapy is less than 20%.Therefore,it has become the urgent problem to be solved that we should clarify the mechanism of 5-Fu resistance,relieve and even reverse the resistance,improve the efficiency of chemotherapy.The annexins are Ca2+-regulated phospholipid-binding proteins widely expressed in all eukaryotic cells except yeasts.The annexins are cytosolic proteins distributed both in the cellular cytoplasm and in the nuclear membranes.They exhibit diverse biological functions by mediating the interactions between proteins in cell membranes with other proteins in cells,in the nuclear membrane and in the extracellular matrix.The annexins have been classified into five groups,A-E.Group A includes 12 members(annexins A1–A11 and A13)found in vertebrates(mammalian).Recent studies have shown that ANXA11 is closely related to the occurrence and development of tumors.Previous research found that the positive expression rate of ANXA11 in gastric carcinoma and the expression intensity was significantly higher than that in normal gastric tissue.The positive expression rate and expression intensity of ANXA11 in lymph metastasis tumor was significantly higher than that of primary gastric cancer.These foundings suggest that ANXA11 is closely related to the occurrence,progression and metastasis of gastric cancer.However,the expression of ANXA11 in different gastric cancer cell lines and its relationship with 5-Fu resistance in gastric cancer have not been reported.In this study,human gastric cancer cell line SGC7901 was used as the research object.RNA interference technology was used to suppress the expression of ANXA11 in SGC7901 cells.Then we detected the sensitivity of different groups to 5-Fu and found that the sensitivity of the transfection group was higher than that of the negative control group and blank control group.At last we had a preliminary discussion of its related mechanisms.It will provide theoretical basis for elucidating the mechanism of 5-Fu resistance in gastric cancer and be of great significance to alleviate and reverse the drug resistance of 5-Fu in gastric cancer and explore new therapeutic targets.Methods: Human gastric cancer cell line SGC7901 and BGC-823 were in cultured.The mRNA and protein expression of ANXA11 in two cell lines were respectively detected by qPCR and Western Blot.ANXA11 high expression cell line SGC7901 was selected.Design and synthese of ANXA11-specific interference siRNA sequence and its negative control sequence,SGC7901 cell line was transiently transfected with Annexin-A11 siRNA and its negative control by means of Lipofectamine 2000.RT-PCR and Western Blot were respectively used to detect ANXA11 mRNA and protein expression after transfection 24h、48h、72h to detected the interference effects.CCK-8 assay was used to detect the sensitivity to 5-Fu by which the IC50 and IC20 values of each group were calculated.Given that the concentration of IC50 in 5-Fu was more lethal to cells,5-Fu of the IC20 concentration was applied to the cells transfected with ANXA11-siRNA-1 in the follow-up experiment.The experimental groups were: ANXA11-siRNA-1 transfection group(hereinafter referred to as transfection group),ANXA11-siRNA-1transfection group combined with IC20 concentration of 5-Fu group(hereinafter referred to as the combined group),negative control group and blank Control group.Apoptosis rate of each group was detected by flow cytometry.QPCR and Western Blot were used to detect the changes of Bax,Bcl-2 and TS mRNA and protein levels in each group.Results:1 The relative expression of ANXA11 mRNA in human gastric cell line SGC7901 and BGC-823 were respectively: 0.045±0.001,0.022±0.004.The relative expression of ANXA11 protein in SGC7901 and BGC-823 cells were respectively1.410±0.180,0.261±0.041.Compared with the BGC-823 cells,ANXA11 mRNA and protein expressions in SGC7901 cells were significantly higher(P=0.033,P=0.000).2 The suppressive effect of ANXA11 was detected by q PCR at mRNA level after transfection with different ANXA11-siRNAs 24,48,72 hours.We found that compared with the negative control group and blank control group,three ANXA11-siRNAs(ANXA11-siRNA-1,ANXA11-siRNA-2,ANXA11-siRNA-3)and the mix of three ANXA11-siRNAs(ANXA11-siRNA123)had different degrees of suppression on the expression of ANXA11,with the suppressive effect of ANXA11-siRNA-1 at 48 h after transfection being the best(F=28.631,P=0.000).SGC7901 cells were transfected with ANXA11-si RNA-1 of different concentrations(20nM,40 nM,60nM,80 nM,100nM).The suppressive effect of ANXA11 was detected by Western Blot at protein level after transfection 24,48,72 hours.We found that compared with the negative control group and blank control group,ANXA11-siRNA-1 of different concentrations could suppress the expression of ANXA11 in different degrees,with the effect of 40 nM concentration at 48 h after transfection being the best(F=24.887,P=0.000).3 In the CCK-8 assay,5-Fu of 1μg/ml,10μg/ml,20μg/ml,40μg/ml,80μg/ml was applied to different groups of SGC7901 cells after transfection24,48,72,96 h.We found that there was significant difference between the optical density of transfection group and negative control group at48h(P<0.05).At this time group,the IC50 of 5-Fu in SGC7901 cells after transfection with ANXA11-siRNA-1 was 6.827±0.770μg/ml,while in negative control group was 11.487±0.669μg/ml,in blank control group was12.053±0.720μg/ml.The corresponding IC20 were 0.086±0.011μg/ml,0.189±0.0153μg/ml.The sensitivity of transfected cells to 5-Fu was significantly higher than that of negative and blank control group(F=47.500,P=0.000;F=45.523,P=0.000).4 The apoptosis rate of SGC7901 cells after transfection was(18.72±2.067)%,while in the combined group was(30.20±2.344)%,in negative control group was(6.66±0.976)%,in blank control group was(6.51±1.509)%.The apoptosis rate increased significantly in the transfection group and the combined group compared with the negative control group and the blank control group(F=118.403,P=0.000),and the apoptosis rate of the combined group was significantly higher than that of the transfection group(P=0.000).5 The expression of ANXA11 protein in SGC7901 cells after transfection with ANXA11-siRNA-1 was 0.523±0.091,while in the combined group was0.167±0.060,in negative control group was 0.887±0.075,in blank control was0.887±0.075.The expression of ANXA11 protein decreased significantly in the transfection and the combined group(F=41.623,P=0.000),and the expression of ANXA11 protein of the combined group was significantly lower than that of the transfection group(P=0.001).6 QPCR and Western was applied to detect the change of mRNA and protein expression of Bax,Bcl-2 and TS after ANXA11-si RNA-1 transfection and transfection combined with 5-Fu.We found that compared with the negative control group and the blank control group,the mRNA and protein expression of Bax in the transfection group and the combined group were up-regulated,while those of Bcl-2 and TS were significantly down-regulated(F=35.608,P=0.000;F=30.048,P=0.000;F=22.874,P=0.000;F=24.396,P=0.000;F=33.890,P=0.000;F=29.630,P=0.000).There was also a significant difference between the transfection group and the combined group(P<0.05).Conclusion:1 ANXA11 expression was higher in gastric cancer cell line SGC7901 than in BGC823.Inhibition of ANXA11 expression can increase the apoptosis rate of gastric cancer SGC7901 cells.2 Suppression of ANXA11 expression can increase the lethality of 5-Fu,and then increase the sensitivity of SGC7901 cells to 5-Fu,and ANXA11 may be a novel gene involved in the drug resistance of gastric cancer cell line SGC7901 to 5-Fu.3 Suppression of ANXA11 expression may be involved in the enhancement of drug sensitivity to 5-Fu of SGC7901 cells by suppressing the expression of TS,down-regulating the anti apoptotic gene Bcl-2 and up-regulating the expression of Pro apoptotic gene Bax. |
