Effect Of Hsa-miR-133a On Breast Cancer Cell Invasion And Migration Potential And Its Mechanism

Background and Objective MicroRNAs are non-coding RNAs of about 22 nucleotides in length that function as negative regulators of gene expression through inhibition of target mRNA translation or degradation of target mRNA to participate in a wide variety of biological processes including cell development, metabolism, proliferation, differentiation and oncogenesis. On the basis of preliminary study which showed that miR-133a expression was significantly reduced in breast cancer and negatively correlated with lymph node metastasis, we designed a study to investigate the effect of miR-133a on breast cancer cell invasion, migration and proliferation potential, and to explore its mechanism.Method(1) Human breast cancer MCF-7 cells were transfected with miR-133a mimics or miR-133a inhibitors by Lipofectamine. MCF-7 cells transfected with negative control (NC) or inhibitor negative control (inhibitor NC) were cultured as negative controls. Cell proliferation potential and invasion potential were evaluated by MTS cell proliferation assay and transwell invasion assay, respectively. Cell migrating ability was detected by transwell migration assay and wound-healing assay.(2) The possible target genes and their functions of miR-133a were forecasted and analyzed by bioinformatics tools.(3) The predicted potential miR-133a target genes associated with invasion, migration and adhesion were selected for this study. After MCF-7 cells were transfected with miR-133a mimics, mRNAs and their proteins expression levels were detected through qRT-PCR and western blot analysis. The 3′UTR region of a target gene FSCN1 was cloned into the psiCHECK-2 vector which containing a luciferase reporter gene and co-transfected with miR-133a mimics into MCF-7, then FSCN1 gene was validated by a luciferase reporter assay.Results(1) Compared with NC group, the cell proliferation inhibition rates were 0.8% (P> 0.05), 8.3%, 8.8% and 18.7% (the latters P<0.05) after MCF-7 cells were transfected with miR-133a mimics for 60h, 84h, 108h and 132h, showing a time-dependent pattern. Cells transfected with miR-133a inhibitors revealed no significantly changes in growth compared to the inhibitor NC transfectant (P>0.05).(2) The cell migration and invasion assay showed that there was a significant reduction in cell migration and invasion capacity after MCF-7 cells transfected with miR-133a mimics for 88h. The number of migrated or invaded cells (10×10 magnification) were 20.4% or 30.3% in miR-133a mimics transfectant compared with the NC transfectant (P<0.01), respectively. While significant cell migration and invasion enhanced were observed after cells were transfected with miR-133a inhibitors for 80h, the number of migrated or invaded cells (10×10 magnification) were 202% or 268% in miR-133a inhibitors transfectant compared with the inhibitor NC transfectant, respectively (P<0.05 and P<0.01).(3) Enhanced cell migration was observed in miR-133a inhibitors transfectant after the scratch (P<0.05).(4) Out of the potential target genes of miR-133a forecasted by bioinformatics tools, some showed their fuctions in promoting cell invasion and migration.(5) MCF-7 cells transfected with miR-133a mimics showed 56% of FSCN1 mRNA expression compared with the control group (P<0.01) and led to a significant decrease in protein expression screened by qRT-PCR and western-blot, respectively.(6) MiR-133a directly binding to FSCN1 3′UTR was confirmed by luciferase reporter assay.Conclusion(1) MiR-133a negatively regulates the invasion and migration potential of breast cancer cells.(2) MiR-133a inhibits breast cancer cell migration and invasion through directly targeting of FSCN1.