Effect and mechanism of miRNA-543 regulating DAZAP2 gene on 5-fluorouracil resistance in gastric cancer cells
Objective To test the expression and prognostic value of microRNA-543(miR-543)in gastric cancer tissues,and explore the impact of miR-543 on 5-fluorouracil(5-FU)resistance of gastric cancer cells,and its underlying mecha-nism.Methods Totally 50 specimens of gastric cancer tissues and cancer-adjacent normal tissues were collected,and the level of miR-543 was tested by quantitative real-time polymerase chain reaction(qRT-PCR).The disease free survival(DFS)of these patients receiving S-l combined with oxaliplatin(SOX chemotherapy)after operation was recorded.Nor-mal gastric epithelial cell line GES-1,gastric cancer cell line AGS and 5-FU resistant cell line AGS/5-FU were cultured,and the expression of miR-543 was detected by qRT-PCR.Moreover,after miR-543 inhibitor was transfected into AGS/5-FU cell line,the sensitivity of gastric cancer cells to 5-FU was detected by MTT assay,and the expressions of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),deleted inazoospermia associated protein 2(DAZAP2),p53,multi-drug resistance related protein 1(MRP1)and thymidylate synthase(TS)were tested in AGS/5-FU cells before and after the transfection by qRT-PCR and Western blot.The target gene of miR-543 was found by bioinformatics analysis and ver-ified by luciferase reporter assay.DAZAP2 rescue experiment was conducted to explore the effect of miR-543 on DAZAP2.Paired t-test was used to compare the expression of miR-543 between gastric cancer tissues and normal tissues.The differences in sensitivity to 5-FU of gastric cancer after inhibiting miR-543 was analyzed by independent sample t-test.Moreover,one-way ANOVA was used to detect the changes of Bcl-2,Bax,DAZAP2,p53,MRP1 and TS lev-els.Results The level of miR-543 was higher in gastric cancer tissues(1.031±0.408)than that in cancer-adjacent nor-mal tissues(0.236±0.056),t=13.640,P＜0.001.Patients with high miR-543 level have shorter DFS than those with low miR-543 level(P=0.008).Results of qRT-PCR demonstrated that the level of miR-543 was higher in AGS/5-FU(7.223±1.260)than those in GES-1(1.306±0.285)and AGS(3.286±1.166),F=16.050,P=0.004,and silencing miR-543 in AGS/5-FU can promote the sensitivity of gastric cancer cells to 5-FU(0.452±0.168),F=12.003,P=0.008.Furthermore,bioinformatics analysis proved that miR-543 targets DAZAP2,and results of qRT-PCR and West-ern blot showed that the expression of Bcl-2 and TS was decreased while the expression of Bax,DAZAP2,and p53 was increased,and MRP1 remained unchanged in miR-543 inhibitor group,which was further proved by luciferase reporter assay.In comparison with cells transfected with miR-543 inhibitor alone,DAZAP2 overexpression+miR-543 inhibitor group can increase the sensitivity to 5-FU and partly reverse the protein levels of Bcl-2,TS,Bax,and p53.Conclusion MiR-543 might exert its function as participating in 5-FU resistance in gastric cancer via DAZAP2.
gastric cancermiR-5435-fluorouracil(5-FU)chemoresistancedeleted inazoospermia associated protein 2(DAZAP2)
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