Targeting Of EIF4G MiR-503 Enhance Drug Sensitivity Of MCF-7/ADR Cells

Objective: In this work, we investigated the potential role of e IF4 G in regulating the expression of ABCB1, ABCC1, ABCG2 proteins, and unravelled the possible association between e IF4 G and miR-503 in the resistance of MCF-7/ADR to anticancer drugs.Methods: Bioinformatics and research software programs were used to predicted the possible miRNA targeted the 3’UTR of e IF4 G. Western blotting tested the expression of p-m TOR, p-4EBP1, ABCB1, ABCC1 and ABCG2 proteins.Quantitative real-time PCR was applied to examine the difference of ABCB1, ABCC1,ABCG2 m RNA. MTT cell proliferation assay was performed to determine cell sensitive of adriamycin, tamoxifen and taxol; the percentage of cell cycle distribution and apoptotic cells was determined by flow cytometry; to validate whether the e IF4 G could be recognized by miR-503, luciferase reporter assay was utilized.Results: The results of bioinformatics software shown that miR-503 and the346-340 bases of e IF4 G 3’UTR gene complementary pairing and there are four binding sites in 327-331 at the same time.The results of western blotting shown ATP-dependent efflux genes(ABCB1,ABCC1, ABCG2) presented a higher protein levels in MCF-7/ADR cells compared to the parental MCF-7 cells. Rapamycin reduced p-m TOR, p-4EBP1, ABCB1, ABCC1 and ABCG2 protein levels at higher(>50 n M) rapamycin doses. It was revealed that miR-503 could cause the downregulation of e IF4 G, ABCB1, ABCC1 and ABCG2.EIF4 G siRNA also negatively regulated the expression of ABCB1, ABCC1, ABCG2.The results of quantitative real-time PCR shown that ATP-dependent efflux genes(ABCB1, ABCC1, ABCG2) presented a higher m RNA levels in MCF-7/ADR cellscompared to the parental MCF-7 cells. MiR-503 and e IF4 G siRNA did not affect the expression of ABCB1, ABCC1 and ABCG2 m RNA while down-regulated e IF4 G in MCF-7/ADR cells.Adriamycin, tamoxifen and taxol showed poor treatment to MCF-7/ADR cells compared with MCF-7 cells by MTT assay. Rapamycin(100 n M) significantly increased the sensitive of 3 drugs, compared with treated with adriamycin, tamoxifen and taxol only. miR-503 could increase the sensitivity of human MCF-7/ADR cells to adriamycin, tamoxifen and taxol, MCF-7/ADR cells show increased sensitive to adriamycin, tamoxifen and taxol downregμLate e IF4 G by siRNA.EIF4G siRNA and miR-503 was identified to be able to block the cell cycle at G0/G1 and lead to the occurrence of apoptosis in MCF-7/ADR cells.Luciferase reporter assay show that the luciferase activity in cells transfected with the vector containing e IF4 G 3’UTR fragment with binding sequence of miR-503 was inhibited by transfection of miR-503 relative to that of the cells that were transfected with negative control miRNA mimics and mock.Conclusion:MiR-503 down-regulated the m RNA and protein level of e IF4 G by targeting e IF4 G directly; e IF4 G has deep relationship with the expression of ABCB1, ABCC1 and ABCG2 transport proteins. miR-503 could increase the sensitivity of human MCF-7/ADR cells to ADM, TAX and TAM may be related to targeting e IF4 G.