Establishment Of Tamoxifen Resistant Breast Cancer Cell Lines And Differential MiRNA Expression Among Taomoxifen Resistant Breast Cancer Cell Lines

Objective To establish human breast cancer tamoxifen resistant cell lines,describe their biological features and explore the potential molecular mechanisms of drug resistance,providing models for the research of tumor resistant mechanism.To screen the differentially expressed miRNAs among tamoxifen resistant and sensitive breast cancer cell lines.Then functional expression of the target genes of those differentially expresses miRNAs was investigated,so as to lay a foundation for further study of the regulatory role of miRNA in drug-resistance.On the basis of the research work,a meta-analysis was performed to comprehensively investigate the relationships between miRNA-21 and clinicopathological features of breast cancer patients.Methods1.The two drug resistant cell lines MCF-7C and MCF-7T were established from the parent MCF-7 cell line by using tamoxifen as a selective drug.MCF-7C cell line was established based on dose stepwise increment exposure to tamoxifen,while MCF-7T was established based on a high concentration of tamoxifen with time stepwise increment exposure.The morphological changes of the cells were observed by microscope.The cell growth curves were plotted using the CCK8 assay and MTT assay was performed to determine the sensitivity of different cell lines to different concentrations of tamoxifen.Moreover,the estrogen receptor(ERa)protein expression was detected by Western blot.2.The HiSeq 2500 sequencing technology and bioinformatics were used to detect the differentially expressed miRNAs in MCF-7 sensitive cell line and tamoxifen resistant cell lines.Then functional expression of the target genes of those differentially expresses miRNAs was investigated.3.On the basis of the research work,a meta-analysis was performed to investigate the relationships between miRNA-21 and clinicoathological features,including estrogen receptor,progesterone receptor,human epidermal growth factor 2,TNM stage and lymph node status of breast cancer patients.Results1.The human breast cancer tamoxifen-resistant cell lines(MCF-7C and MCF-7T)were established and can grow steadily in a culture medium containing 10 μM tamoxifen.The different morphological changes of the cell lines were observed by microscope.The results of CCK-8 assay showed that MCF-7C and MCF-7T cell lines exhibited significantly greater proliferation than MCF-7 cell lines in vitro.MTT results showed that,at the same concentration effect of TAM,tamoxifen-resistant cell lines(MCF-7C and MCF-7T)exhibited greater cell variability than MCF-7 cell.Western blot assay showed that the expression of ERa protein was increased in tamoxifen-resistant cell lines(MCF-7C and MCF-7T).2.A total of 1,646 miRNAs were predicted,with 1,376 known miRNAs and 270 newly predicted miRNAs.162 miRNAs were differentially expressed between MCF-7C and MCF-7 cell lines,62 miRNAs were differentially expressed between MCF-7T and MCF-7 cell lines,and 175 miRNAs were differentially expressed between MCF-7C cell and MCF-7T cell lines.The function classification of GO,and COG and KEGG metabolic signal pathway analysis between differentially expressed miRNAs were almost consistent,indicating that the growth of drug-resistant tumor cells was closely related to the cell metabolism and the regulation of related functional proteins.3.A total of 11 relevant articles were included in this meta-analysis.The overall results showed that miR-21 overexpression was associated with positive HER-2 expression and advanced TNM stage as well as lymph node metastasis.Subgroup analysis revealed significant associations between miR-21 expression and hormone receptor status,and the associations varied in Chinese and other populations.Conclusion1.The establishment of the tamoxifen resistant breast cancer cell lines(MCF-7C and MCF-7T)can be used to further investigate the mechanisms of tamoxifen resistance.2.miRNAs were differentially expressed between tamoxifen-resistant cell lines and sensitive cell lines,suggesting that miRNAs could play an important role in the formation of TAM resistance in breast cancer.3.Overexpression of miR-21 was associated with hormone receptor,HER-2,TNM stage and lymphy node metastasis of breast cancer patients,suggesting that miR-21 could serve as a promising biomarker of an aggressive breast cancer phenotype and a novel tumor therapeutic target of breast cancer.