Investigating The Effects Of Cell Growth And RUNX3 Gene Silence Induced With ShRNA In SGC7901 Cell Line

Objective To observe the characteristics of human stomach carcinoma cell line SGC7901 after the transcriptional gene silencing of RUNX3 induced by short hairpin RNAs targeting to prompter regions of it,and to analyze the suppress- tumor mechanism of RUNX3 in the occurrence and development of stomach cancer.Methods According to the principle of RNAi design,pSilencer3.1-H1-shRNA /RUNX3 expression vector was constructed,and the recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 by liposome. Following, the positive transfected cell clones were screened with G418. In order to elucidate the effect of expression of RUNX3 gene in the stable transfected cell line SGC7901,its mRNA and protein expression level were determined by RT-PCR,Western blotting and immunocytochemistry methods. And the characteristics of the cell line SGC7901, pSilencer3.1/ SGC7901 andp Silencer3.1-H1-shRNA /RUNX3/ SGC7901 were analyzed with growth curves, clone formation rate,cell cycle distribution as well as HE staining after RUNX3 gene loss-regμlated expression.At last,pSilencer3.1-H1-shRNA /RUNX3/ SGC7901 was treated with the different concentration of 5'-Aza-2'-deoxycytidine,and the activating level of RUNX3 gene was examined by RT-PCR and Western blotting.Resμlts The resμlts of restriction enzyme digestion and DNA sequencing demonstrate that the pSilencer3.1-H1-shRNA/RUNX3 eukaryotic expression vector was successfμlly constructed. The pSilencer3.1-H1 and pSilencer3.1-H1-shRNA/ RUNX3 were transfect into SGC7901 cells by Lipofectamine 2000 respectively. the positive cell clones of pSilencer3.1-H1-shRNA/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were selected by G418 and by identification of RT-PCR,western blotting and immunocytochemistry,the pSilencer3.1-H1-/SGC7901 cell line and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 cell line were established successfμlly. The cell growth rate of pSilencer3.1-H1-shRNA/RUNX3/SGC7901 was fastest than those transfected with the pSilencer3.1-H1 and the untransfected SGC7901 (P<0.05) through the cell growth curve. The colony formation in soft agar displayed that colony formation efficiency of pSilencer3.1-H1-shRNA/RUNX3/SGC7901 was (17.4±0.31)%,but the colony formation efficiency of pSilencer3.1-H1/SGC7901 and SGC7901 were (9.9±0.3)% and (9.8±2.1)% respectively. The colony formation ratio of pSilencer3.1-H1-shRNA/RUNX3/SGC7901 was increased in comparison with that of pSilencer3.1-H1/SGC7901 and SGC7901 (P<0.01). To further investigate the change of cell cycle after sclencing RUNX3 gene,flow cytometry(FCM) showed that the cell distribution in G0/G1 and S phrase of pSilencer3.1-H1-shRNA/RUNX3/SGC7901 was lowest and highest respectively(P<0.05),compared with that of the transfected pSilencer3.1-H1 and untransfected cells.And HE staining indicated that the patho-caryocinesia and the tumor giant cells in pSilencer3.1-H1-shRNA/RUNX3/ SGC7901 was the most compared with that of the transfected pSilencer3.1-H1 and untransfected cells. At last, through the ananlysis of RT-PCR and western blotting assay,the inactivation of RUNX3 was not reactivate with 5'-Aza-2'-deoxycytidine.Conclusion1. The eukaryotic expression vector of pSilencer3.1-H1-shRNA/RUNX3 were constructed.And the pSilencer3.1-H1-shRNA/RUNX3/SGC7901 which silencing RUNX3 gene was established successfμlly.2. After loss-expression of RUNX3 protein in SGC7901 cells, the data showed that the proliferation speed of cells was high, the colony formation rate in soft agar was enhanced,the cell distribution in G0/G1 and S phrase was lowe and high respectivelyas well as patho-caryocinesia and the tumor giant cells was increased.3. After treatment the pSilencer3.1-H1-shRNA/RUNX3 /SGC7901 cells with 5'-Aza-2'-deoxycytidine, the activity of silcencing RUNX3 by shRNA did not return.