Effect And Mechanism Of Let-7i On Chemotherapy Drug Resistance Of Breast Cancer Cells

Objective: To investigate the difference of expression of miRNA let-7i in breast cancer parental and drug-resistant cells and its correlation with breast cancer chemotherapy resistance.To investigate the effect of let-7i level changes on chemotherapy drug sensitivity of breast cancer-resistant cells.To investigate the effect of let-7i level changes on the function of chemotherapy-induced apoptosis of drug-resistant breast cancer cells.To investigate the effect of let-7i level on the downstream possible target gene K-RAS and related apoptotic proteins in breast cancer cells,and to explore the function of let-7i on chemosensitivity of breast cancer cells and possible mechanism.Methods:1.Reverse transcription-Polymerase Chain Reaction(RT-PCR)was used to verify the expression of miRNA let-7i in different drug resistant cell lines and corresponding parent breast cancer cells,and to verify the correlation between the expression of let-7i and drug resistance of breast cancer cells.2.Overexpression and inhibition of let-7i expression in drug-resistant cell lines were carried out by liposome transfection.The IC50 of Adriamycin(ADR)was detected by CCK-8 to verify the effect of changes of let-7i level on drug resistance of drug-resistant breast cancer cells.3.The effect of let-7i level changes on the chemotherapy sensitivity of breast cancer resistant cells to ADR was evaluated by a plate clone assay.4.Flow cytometry and caspase3/9 activity were used to evaluate the apoptosis-inducing effect of the changes of let-7i level in drug-resistant cells.5.RT-PCR and western-blot were used to detect the effect of changes of let-7i level on possible target genes and related apoptotic proteins.Results: 1.The effect of let-7i level on chemotherapy-drug sensitivity in drug-resistant breast cancer cells.(1)Compared with breast cancer parental cells(MCF-7),the expression of let-7i was significantly down-regulated in breast cancer resistant cells(MCF-7/ADR and MCF-7/DDP).(2)The chemotherapy sensitivity of MCF-7 / ADR and MCF-7/DDP cells to Adriamycin were increased after overexpression of let-7i in MCF-7/ADR and MCF-7/DDP cell line,and the chemotherapy sensitivity of MCF-7/ADR and MCF-7/DDP cells to Adriamycin were decreased after inhibiting the expression of let-7i.(3)Plate clone formation assay showed that the overexpression of let-7i,at the same concentration of chemotherapeutic drugs,could inhibit the plate-forming ability of MCF-7/ADR and MCF-7/DDP cells,while the inhibition of let-7i expression could promote the plate-forming ability of MCF-7/ADR and MCF-7/DDP cells.2.The effects of let-7i level on apoptosis function of drug-resistant breast cancer cells.(1)The results of flow cytometry showed that the overexpression of let-7i could inhibit the apoptosis of MCF-7/ADR and MCF-7/DDP cells at the same Adriamycin concentration.(2)The results of caspase 3/9 activity assay showed that the overexpression of let-7i increased the caspase 3/9 activity of MCF-7/ADR and MCF-7/DDP cells at the same Adriamycin concentration.(3)Western-blot results showed that the level of pro-apoptotic protein Bax increased and the level of anti-apoptotic protein Bcl-2 decreased with the increase of Adriamycin concentration gradient.3.The effects of let-7i level changes on downstream target gene k-ras and related apoptotic proteins in MCF-7/ADR and MCF-7/DDP cells.(1)The expression of and K-RAS genes in MCF-7/ADR and MCF-7/DDP cells was significantly higher than that in breast cancer parental cells.The expression of Bcl-2 was also increased in MCF-7/ADR and MCF-7/DDP cells compared with MCF-7 cells.(2)RT-PCR and western-blot results showed that the expression levels of Bcl-2 and K-RAS were reduced not only at the mRNA level,but also at the protein level after the expression level of let-7i was overexpressed in MCF-7/ADR and MCF-7/DDP cells.Conclusion: In drug-resistant breast cancer cells,the expression of miRNA let-7i is negatively correlated with chemotherapy resistance of breast cancer cells.Overexpression of let-7i level can increase the chemo-sensitivity of drug-resistant breast cancer cells and promote the apoptotic function of drug-resistant breast cancer cells.The chemo-sensitivity of breast cancer resistant cells may be affected by regulating the K-RAS and Bcl-2/Bax pathways.