Study Of Cell Secreted MiRNA Regulating Migration And Invasion Of Breast Cancer Cell BT549

MiRNAs are a class of non-coding small molecule RNA,which are 19-25 nucleotides long.Through complete or partial complementation to miRNA recognition elements on 3' untranslated region of the target mRNA,they degrade target mRNA or inhibit its translation to regulate a series of important life processes.MiRNAs can not only play a role in the regulation of gene expression inside cells,but also affect the biological activity of other cells in the microenvironment in vivo through the way of cell secretion.However,in the microenvironment of breast cancer,whether the heterogenous breast cancer cells can be mutually regulated by the form of cell secreted extracellular miRNA has not been reported.Objective:To investigate the effects of cell secreted miR-200c?miR-205 on the migration and invasion activities of BT549 cells.Methods:1.Screening miRNA associated with breast cancer metastasis from high throughput miRNA expression profiling data: Searching for miRNA expression microarray profile associated with breast cancer metastasis in the GEO database;Using GEO 2R software for data analysis,to obtain the differentially expressed miRNA in two groups of breast cancer cells with different malignancy;Screening microRNA as the experimental object.2.Effects of miR-200 c,miR-205 on migration and invasion activities of breast cancer cell line BT549: hsa-mir-200 c and hsa-miR-205 gene packaged lentivirus were harvested to infect breast cancer cell BT549;Positive clones of infected cells were sorted out by puromycin(PM)including stable miR-200 c over-expressed BT549 cell(BT549 miR-200c)and stable miR-205 expressed BT549 cell(BT549 miR-205).qRT-PCR assay tested the correspondent miRNA expression in cell lines of BT549 miR-200 c and BT549 miR-205;Transwell migration assay was applied to detect the cell migration ability;Transwell invasion assay was employed to analyze the cell invasion ability.3.Effect of cell secreted miR-200 c,miR-205 on migration and invasion activities of breast cancer cell line BT549: Cultured medium from stable over-expressed miR-200 c,miR-205 breast cancer cell BT549 was mixed at 1:1 ratio with normal cell culture medium;Wild-type BT549 were incubated with the mixed medium for 96 hours;qRT-PCR assay tested the miR-200 c,miR-205 expression in wild-type BT549;Transwell migration and invasion assays were applied to detect the migration and invation ability of wild-type BT549.Results:1.GSE62022 and GSE40056 datasets from the GEO database were enrolled for subsequent analysis;GEO 2R was used to obtain differentially expressed miRNA associated with metastasis of breast cancer;PROGmiR and BreastMark secondary database show that higher expression of miR-200 c,miR-205 in breast cancer patients correalates with a higher survival rate.2.Puromycin sorted lentivirus infected BT549 cells exihibited obvious green fluorescence;qRT-PCR showed that expression of miR-200 c,miR-205 are increased in BT549 miR-200 c and BT549 miR-205 cell lines compare to the BT549 control(P< 0.05);Transwell migration test showed decreased migration ability for BT549 miR-200 c and BT549 miR-205 cell lines;Transwell invasion assay showed reduced invasion ability for BT549 miR-200 c and BT549 miR-205 cell lines.3.qRT-PCR showed increased levels of miR-200 c,miR-205 secreted from breast cancer cells which had stable over-expressed of miR-200 c,miR-205(P<0.05);Transwell migration and invasion assays showed that compared to control group,the migration and invasion ability are decreased in BT549 cultured with mixed medium from BT549 miR-200 c,BT549 miR-205(P<0.05).Conclusions:1.BT549 cell lines with stable over-expressed miR-200 c,miR-205 were successfully constructed;miR-200 c,miR-205 can inhibit the migration and invasion abilities of breast cancer cell line BT549 in vitro.2.Increased level of secreted extracellular miR-200 c,miR-205 were detected from culture medium of BT549 cell lines with stable over-expressed miR-200 c,miR-205.3?Cell secreted miR-200 c,miR-205 can reduce the migration and invasion abilities of breast cancer cell line BT549.