| Part Ⅰ Expression and significance of miR-16 in breast cancer tissues and cells Objective:In the early stage,our research group screened the difference of miRNAs level between adriamycin resistant and sensitive breast cancer cell lines.MiR-16 was found to be highly expressed in adriamycin resistant breast cancer cell lines.On the basis of previous studies,we intend to explore the expression level of miR-16 in breast cancer cell lines and tissues and its relationship with clinical pathological indicators and prognosis of breast cancer patients.The correlation between miR-16 expression level and adriamycin resistance in breast cancer and its impact on biological function of breast cancer were also analyzed.Methods:1.Parental breast cancer cell line(MCF-7 /S)and its adriamycin-resistant cell line(MCF-7 /Adr)were used in the study.The expression of miR-16 in these two cell lines was verified by real-time quantitative PCR(RT-qPCR).2.The expression of miR-16 in the same cell line before and after transfection with miR-16 mimics and inhibitor was detected by RT-qPCR.MTT assay and flow cytometry were used to detect the effect of miR-16 expression changes on adriamycin resistance in breast cancer cells.3.65 cases of breast cancer receiving neoadjuvant chemotherapy in the first affiliated hospital of nanjing medical university from September 2012 to February 2016 were collected,and 62 breast cancer formalin fixed paraffin embedded tissues that met the research criteria were obtained.This study was approved by the ethics committee of nanjing medical university,and all patients provided written informed consent.Resistance to adriamycin is defined as nonresponse to treatment(stabilizationor progression)or recurrence as adjuvant therapy within 6 months of discontinuation of treatment.A total of 40 cases of BC(20 cases of adriamycin resistance and 20 cases of no adriamycin resistance)participated in the study,and the expression levels of miR-16 in the effective group and the ineffective group after neoadjuvant chemotherapy were compared.The expression of miR-16 in the tumor tissues of the above patients with breast cancer was detected by RT-qPCR,and the correlation between miR-16 and drug resistance of breast cancer was analyzed.The relationship between miR-16 expression level and clinical pathological indicators in breast cancer patients4.42 breast cancer patients with complete follow-up data were selected to analyze the relationship between miR-16 expression level and prognosis.the predictive value of mir-16 for the prognosis of breast cancer patients was explored.Results:1.RT-qPCR results showed that compared with MCF-7 /S,miR-16 was lower expressed in MCF-7 /Adr,which was consistent with the results of the chip at the early stage.2.Compared with the negative transfection control,after the transfection of MCF-7/Adr with miR-16 inhibitors,the expression of miR-16 was significantly decreased,cell IC50 was also significantly increased,and cell apoptosis was decreased.After the mimics transfection,,the expression of miR-16 was significantly increased,cell IC50 was significantly decreased,and apoptosis was increased.3.The tumor tissues of breast cancer patients receiving neoadjuvant chemotherapy were detected.Compared with the specimens in the neoadjuvant chemotherapy effective group,miR-16 was decreased in the ineffective group.The expression of miR-16 was correlated with tumor size and TNM stage4.Follow-up data confirmed that the disease-free survival rate of patients in the miR-16 high-expression group was significantly lower than that of miR-16 patients in the low expression group.Conclusion:The expression of miR-16 in drug-resistant breast cancer cells(MCF-7 /Adr)was significantly lower than that in sensitive breast cancer cells(MCF-7/S)and low expression in breast cancer tissues after neoadjuvant chemotherapy.After in vitro transfection of miR-16 mimics and inhibitors,it was found that the expression level of miR-16 was negatively correlated with the drug resistance of breast cancer cells to adriamycin(Adr).MiR-16 is negatively correlated with tumor size and TNM stage,and the low expression of miR-16 is associated with poor prognosis of breast cancer patients.Therefore,miR-16 may become a new target for the diagnosis and treatment of breast cancer and predict the prognosis of breast cancer patients..Part Ⅱ MiR-16 targets the regulation of wip-1 /Bcl-2 in adriamycin resistance in breast cancer Objective:The target genes and downstream pathways regulated by miR-16 were identified and verified to further investigate the effect of miR-16 on adriamycin resistance in breast cancer cells and its potential mechanism.Methods:1.MiRWalk was used to predicted the possible target genes and the target genes were selected through preliminary experiments and literatures in the gene and protein level.Further evidence of direct interaction between the target gene and miR-16 was provided through dual-luciferase reporting assay,and the binding sites were determined.The expression of Wip-1 and Bcl-2 in breast cancer cell lines before and after transfection was detected.2.MRNA and protein levels of Wip-1 and l-2 in breast cancer tissues and cells of each group were detected by RT-qPCR and Western blot,respectively.Immunofluorescence was used to detect the subcellular localization of Wip-1 and Bcl-2 in cytoplasm and nucleus.Results:MiRWalk predicted the target genes of miR-16 and conducted pathway enrichment analysis,which was found that miR-16 was enriched in the Wip-1 and Bcl-2 pathways.MiR-16 inhibits the expression of Wip-1 and Bcl-2 by targeting the 3’UTR of PPM1 D and Bcl-2.In the presence of miR-16,the luciferase activity of the constructs containing wild-type PPM1 D and Bcl-2 3’UTR was significantly reduced by 75% and 55%,respectively.However,there was no difference in cell transfection of the mutants PPM1 D and Bcl-2 3 ’UTR,suggesting that PPM1 D and Bcl-2 genes were direct targets of miR-16.In breast cancer tumor tissues and breast cancer cells,the level of miR-16 expression was negatively correlated with the level of Wip-1 and Bcl-2 Western blotting and immunofluorescence were used to detect the expression of Wip-1 and Bcl-2 in MCF-7 /S and MCF-7 /A cells.The results showed that the protein expressions of Wip-1 and Bcl-2 were increased when the expression of miR-16 was decreased in MCF-7 /A cells.Conclusion:MiR-16 can regulate the expression of Wip-1 and Bcl-2 in MCF-7 /Adr and MCF-7 /S cells.Adriamycin resistance can be partially affected by regulating the Wip-1 and Bcl-2 pathways,which may be an important molecular mechanism for adriamycin resistance in breast cancer cells. |
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