The Effects Of ShRNA Targeting Silence Of YB-1 Gene On The Malignant Biological Behaviors Of Breast Cancer MDA-MB-231 Cells

Objective To construct the expression vectors of siRNA targeting the YB-1 gene and identify its inhibition efficiency. To observe the effects of targeting silence of Y-box binding protein 1 (YB-1) gene on the malignant biological behaviors of breast cancer MDA-MB-231 cells.Methods Three short hairpin siRNAs targeting YB-1 gene were designed and chemically synthesized,after annealing to form double-strand DNA and inserting into plasmid pGenesil-1 whose segments were digested by enzyme, then enzyme digestion analysis and DNA sequencing were performed.The identified recombinant YB-1 shRNA plasmids were transfected into MDA-MB-231cells with liposome transfection method. The expressions of YB-1 gene at mRNA and protein levels were identified by fluorescent quantitative PCR and Western blot, respectively. The recombinant plasmid of YB-1 short-hairpin interference RNA (shRNA) which was most effective on inhibiting the expressions of YB-1 gene in MDA-MB-231 cells was constructed and transfected into human breast cancer MDA-MB-231 cells by lipofectamine mediation. The expressions of YB-1, MMP-2 and MMP-9 genes at mRNA and protein levels were determined by RT-PCR and Western blot respectively, and the changes in cell proliferation and apoptosis were assessed by MTT assay and flow cytometry, respectively , and the Transwell test was used to detect the invasion ability of MDA-MB-231 cells.Results Sequencing proved that recombinant plasmids pGSi1, pGSi2 and pGSi3 were constructed correctly, and of the three recombinant plasmids, pGSi2 was screened as most effective YB-1 shRNA recombinant expression vector in MDA-MB-231 cells. The expressions of YB-1 mRNA and protein in the cells transfected with pGSi2 was much less than that of the cells transfected with negative contrasting HK (P<0.05).The silencing of YB-1 could inhibit the proliferation ability and induce the apoptosis of MDA-MB-231 cells as well as the migration capability (all P<0.05), and the expressions of MMP-2 and MMP-9 were significantly reduced than the HK groups (all P<0.05).Conclusions The expression vectors of siRNA targeting the YB-1 gene have been successfully constructed, and an siRNA with significant inhibitory efficiency targeting YB-1 gene is screened. YB-1 knockdown significantly inhibits the proliferation and promotes apoptosis,as well as inhibits the invasive ability, the expressions of MMP-2 and MMP-9 in MDA-MB-231 cells.