Effect of RNA interference of Gli1 gene expression on proliferation and apoptosis of glioma cell line U251

Objective To investigate the inhibitory effect of RNA interference （RNAi） on Glil, Bcl-2, Box and cycin DI gene expressions in U251 cell line and the proliferation of U251 cells. Methods Small interfering RNA （siRNA, at locus of 58, 59, 60 and 61） targeted for Glil gene was designed and transfected into U251 cells. RT-PCR was emplyed to detect the mRNA expression of Glil gene to select the siRNA interference fragment （siRNA-Glil） that could most efficiently inhibit the mRNA expression of Glil gene. The mRNA and protein expressions of Glil gene at different times after siRNA-Glil transfection were detected to determine the time law of this interference. U251 cells at logarithmic phase were divided into 3 groups： siRNA-Glil group （transfection of selected siRNA-Glil fragments）, siRNA-NC （transfection of siRNA fragments） and siRNA-N group （blank controls）. The mRNA and protein expressions of Bcl-2, Box and cycin D1 gene were assessed by RT-PCR and Western blotting. Proliferation of cells was measured by MTT assay, and cell apoptosis and cell cycles were detected by flow cytometry （FCM）. Results Transfection efficiency of interference fragments （at locus of 58, 59, 60 and 61, and NC） reached 69.2%; RT-PCR indicated that no obvious Glil mRNA expression was noted at U251-60 cells 48 h after the transfection, therefore, locus 60 was the best interference fi＇agment and 48 h was the best time. The mRNA and protein expressions of Bcl-2 and cycin D1 genes were obviously suppressed by siRNA, and the rnRNA and protein expressions of Bax gene were significantly up-regulated in the siRNA-Glil group as compared with those in the siRNA-N and siRNA-NC groups 48 h after transfection （P〈0.05）. Silencing Glil by RNAi significantly inhibited the proliferation and induced the apoptosis of U251 cells as compared with siRNA-N and siRNA-NC groups 24, 48 and 72 h after transfection （P〈0.05）. Cells at GO and G1 phases were obviously increased and those at S phase were significantly decreased in the siRNA-Glil group as compared with those in the siRNA-N and siRNA-NC groups （P〈0.05）. Conclusion Expression of Glil gene can be effectively inhibited by specific siRNA targeting Gill gene in U251 cells and the proliferation of U251 cells can be significantly inhibited, which may possibly be related to that siRNA-Glil decreases the expressions of Bcl-2 and cycin D1 and alters the ratio of Bcl-2/Bax. Key words: Hedgehog signal way;  Signal transduction;  RNA interference