| Objective:To investigate the expression of Y-box binding protein-1(YB-1) in human breast carcinoma and analyze the relation between YB-1 and the metastasis of the cancer and other clinical parameters; The changes of the mobility, invasiveness and migration capability of the breast cancer cells were evaluated after the YB-1 expression was downregulated by YB-1shRNA.Method:Firstly, immunohistochemistry(SP kit) was used to detect the expressions of Y-box binding protein-1 (YB-1) in 50 samples of human breast carcinoma, and then the relation between the expressions of YB-1 and clinical parameters as well as the expressions of ER, PR and Her-2 were analyzed. Then, the recombinant eukaryotic expression plasmid including anti-YB-1 gene and the vector-random-sequence were introduced into human breast cancer cells (MCF-7) by lipofectamine mediation and positive clones were screened by G418. RT-PCR and Western blotting were employed to detect the expression of YB-1 mRNA and protein in MCF-7 cells. A wound healing assay and transwell chamber models in vitro were used to detect the mobility, invasiveness and migration capability of cells. Meanwhile, RT-PCR, Western blotting and Gelatin Zymography were used to detect the expression levels of MMP-2mRNA, MMP-2 protein and the activity of MMP-2 protein separately.Results:①Among the 50 cases paraffin-embedded specimens of human breast carcinoma, there were 30 cases had YB-1 nuclear expression while all the cases had YB-1 endochylema expression. No correlation was found between the YB-1 nuclear expression and some clinical parameters, including age (P=0.529), tumor size (P=0.917), clinical stage (P=1.000)and pathological type (P=0.167), while YB-1 nuclear expression had a correlation with the lymph node metastasis(P=0.001, r=0.523). Meanwhile, YB-1 nuclear expression was correlated with ER (P=0.004, r=-0.417), PR (P=0.002, r=-0.442) and Her-2 (P=0.021, r=0.361).②The recombinant plamids pGensil-1.1/HK, pGensil-1.1/YBX11, pGensil-1.1/YBX12 and pGensil-1.1/YBX13 were transfected into the human breast cancer cell line MCF-7, then four cell lines were obtained as followed:MCF-7/HK, MCF-7/Y1, MCF-7/Y2 and MCF-7/Y3, positive clones had been screened by G418 for 2 weeks. The expression of YB-1mRNA in MCF-7/Y1, MCF-7/Y2 and MCF-7/Y3 cell lines were much lower than MCF-7 and MCF-7/HK cell lines. Furthermore, the expression of YB-1mRNA in MCF-7/Y2 was significantly lower compared to the other two cell lines(P＜0.05). So the MCF-7/Y2 was chosen for further research and it was named as MCF-7/YB-1 while the MCF-7 and MCF-7/HK were used as blank and negative control separately. Western blotting showed that the expression of YB-1 protein in MCF-7/YB-1 was much lower than the two control groups. The mobility, invasiveness, migration capability of the MCF-7/YB-1 were much lower than the two control groups while the differences between the two control groups were not significant. Furthermore, the expression of MMP-2mRNA in MCF-7/YB-1 was significant lower than two control groups (P＜0.05) while the difference between the two control groups was not obvious. Parallel to this, the expression of MMP-2 protein in MCF-7/YB-1 was much lower than other two groups as well as the activity of the protein.Conclusion:①YB-1 nuclear expression in human breast carcinoma had no significant correlation with age, tumor size, clinical stage and pathological type, but it had a positive correlation with the lymph node metastasis. Meanwhile, YB-1 nuclear expression was negatively correlated with ER and PR while it was positively correlated with Her-2, so YB-1 nuclear expression was a poor predictor of prognosis.②Transfection with YB-1shRNA gene could inhibit the expression of YB-1mRNA and protein. As followed, the mobility, invasiveness, migration capability of the cells were decreased as well as the expression of MMP-2mRNA and protein while the activity of MMP-2 protein was also decreased. It predicted that YB-1 would effect the mobility, invasiveness, migration capability of cells through the regulation of MMP-2. To sum up, YB-1 had a correlation with the migration and invasison of human breast carcinoma, and a new strategy targeting to YB-1 maybe a new effective method for the therapy of human malignancies. |
PDF Full Text Request