Construction and functional characterization of alentiviralvector mediating RNA interference of DEK gene / 军事医学

Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.

Effect of phosphatase activity of PTEN on migration ability of human breast cancer cell line ZR-75-1 / 肿瘤

Objective: This study was to explore the effects of phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10) on migration ability of human breast cancer ZR-75-1 cells. Methods: Three kinds of plasmids, wt-PTEN plasmid, G129R-PTEN plasmid, and G129E-PTEN plasmid, were constructed and transfected into human breast cancer cell line ZR-75-1, with a deletion of PTEN gene, via liposome mediation. The expression of PTEN protein as well as the Tyr397 in phospho-focal adhesion kinase (p397.FAK) was measured by Western blotting. The effect of phosphatase activity of PTEN was observed in a cell scratch wound model in vitro. The inhibitory effects of phosphatase activity of PTEN were measured by using cell-matrix adhesion test and reconstituted basement membrane invasion technique. The expression level of MMP-2 was determined by immunohistochemical staining. Results: Three kinds of plasmids (wt-PTEN, G129R-PTEN, and G129E-PTEN) were successfully transfected into ZR-75-1 cells, respectively. All the three stably transfected cells had PTEN protein expression. Transfection of wt-PTEN and G129R-PTEN inhibited the migration of ZR-75-1 cells. There was significant difference in the inhibitory degree of cell adhesion and invasion between wt-PTEN or G129E-PTEN groups and G129R-PTEN transfected or non transfected groups (P < 0.01). The level of p397.FAK was significantly lower in wt-PTEN and G129E-PTEN groups compared with G129R-PTEN transfected groups. There was significant difference in the expression level of MMP-2 between G129R-PTEN-transfected and non transfected groups(P < 0.01). Conclusion: Both wild-type PTEN with dual specific phosphatase activities and G129E-PTEN with only protein phosphatase activity have inhibitory effects on migration of human breast cancer cell line ZR-75-1. However G129R-PTEN without phosphatase activity have no effects.