RESUMO
Objective To investigate the effects of c?myc promoter binding protein 1(MBP?1)gene on the proliferation of human Saos?2 osteo?sarcoma cells in vitro. Methods Saos?2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP?1 shRNA). Two MBP?1 shRNA sequences and one neg?ative control shRNA sequence were designed ,synthesized and cloned into pSIREN?retroQ plasma. Then the recombinant plasmids were construct?ed and transfected into human Saos?2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP?1 mRNA and protein in Saos?2 cells were detected by real?time PCR and Western blot ,respectively. The effects of altered expression of MBP?1 on cell proliferation were measured by CCK?8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos?2 were determined by Western blot. Results PCR and sequenc?ing results indicated that the recombinant plasmids pSIREN?retroQ was constructed. The relative expression level of MBP?1 mRNA in the MBP?1 siRNA transfection group was significantly decreased than that in blank control group(P<0.05). Compared with the blank control group,the ex?pression levels of MBP?1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos?2 cells at 48,72,and 96 hours after MBP?1 siRNA transfection were significantly increased than those in the blank control group(P<0.05). Compared with the blank con?trol group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P<0.05). Conclusion Knockdown of the expression of MBP?1 gene promotes the proliferation of human Saos?2 osteosarcoma cells. MBP?1 gene may become the new tar?get of gene therapy for osteosarcoma.
RESUMO
Objective To silence human gene Set7/9 and screen out stable transfection cell line in hepatocellular carcinoma cell line HepG2 so as to investigate the impact of down-regulation of Set7/9 in cell line HepG2 and provide experimental foundation for studies on the effect of set7/9 in HepG2.Methods The target oligo was designed and synthesized;shRNA interference vector and the control vector were constructed and transfected into HepG2 cells;the stable transfection cells were screened out.Then Real-time PCR and Western blot were performed to detect the silence of Set7/9 according to both gene expression and protein expression level. Results The shRNA interference vector was constructed and transfected into HepG2 cells successfully.Compared with that in the negative control group,the expression of Set7/9 was dramatically downregulated (P < 0.05 ). Meanwhile,the expression of related protein Sirt1 and Suv39h1 was upregulated 8.4 folds and 1.1 fold, respectively.Conclusion Downregulation of Set7/9 expression can upregulate Sirt1 and Suv39h1,suggesting that Set7/9 may affect the activity of HepG2 cell lines.