Effects Of Microrna137 Targeting YB-1 Gene On The Malignant Behaviors Of Human Breast Cancer MDA-MB231 Cell

Objective To investigate the inhibition effects of microRNA targeting Y-box binding protein 1(YB-1) gene on the malignant behaviors of human breast cancer cell MB-MDA231.Methods A series of bioinformatics analysis were performed to predict possible microRNA targeting the 3'UTR of YB-1 gene. According to the results from bioinformatics analysis, the microRNA overexpression pGensil1-pre-microRNA plasmids were constructed. The vectors were transfected into MDA-MB-231 cells with lipofectamine, and the expressions of microRNAs were detected by real-time PCR. Then the expressions of YB-1 mRNA and protein were analyzed by real-time PCR and Western blot respectively. To evaluate the relationship of microRNA with 3'UTR of YB-1 gene, the dual luciferase assays were performed. The monoclonal cell lines that overexpressing microRNA with best inhibitory effect selected by G418. MTT assays were used to observe the inhibitory effect of microRNA on cell proliferation in vitro, and FCM were used to detect the cell cycle and apoptosis, meanwhile, the effects on cell invasion in vitro were detected by Transwell tests. The expressions of MMP2 and MMP9 also were detected to observe this feature in cells.Results The possible microRNAs targeting the 3 UTR of YB-1 gene were predicted by bioinformatics analysis softswares. According to the results from bioinformatics analysis, the microRNA overexpression pGensil1-pre-mir216/137/153 plasmids were constructed, and identified by enzyme restriction digestion and sequence analysis. pGensil1-pre-mir137 with best inhibitory effect was detected by RT-PCR,real-time PCR and western blot, and the monoclonal cell lines selected by G418. Compared with control groups, the expression of microRNA137 was increased remarkably (P<0.05), the expression of YB-1 mRNA and protein level were significantly reduced (P<0.05). Luciferase reporter activities reduced by 30% compared with control groups. The growth ability of cells in experimental group were lower than that of control groups (P<0.05). Compared with control groups, the percentage of cells in the phage of G1 was increased significantly (P<0.05) and that in the phase of S was decreased remarkably (P<0.05), and the rate of apoptosis was increased remarkably (P<0.05). The abilities of invasion were decreased (P<0.05) in experimental group, and the mRNA and protein levels of MMP2 and MMP9 genes were also decreased (P<0.05).Conclusions The microRNA137 overexpression plasmid is successfully constructed. The monoclonal cell lines which overexpressing microRNA137 are successfully selected. MicroRNA137 could inhibit cell proliferation, cell cycle progression and invasion of MB-MDA231 cells by directly targeting YB-1.