Effect of RNA interference of Gli1 gene expression on proliferation and apoptosis of glioma cell line U251
10.3760/cma.j.issn.1671-8925.2011.08.004
- VernacularTitle:RNAi沉默Gli1基因对人胶质瘤细胞U251增殖与凋亡的影响
- Author:
Hai-Long TIAN
1
;
Jing-Ping BAI
;
Hui-Wu LI
;
Hui LI
;
Chen LIU
;
Ming-Jun DUAN
Author Information
1. 新疆医科大学附属肿瘤医院
- Keywords:
Hedgehog signal way;
Signal transduction;
RNA interference
- From:
Chinese Journal of Neuromedicine
2011;10(8):768-773
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the inhibitory effect of RNA interference (RNAi) on Gli1,Bcl-2, Bax and cycin D1 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 Gli1 gene was designed and transfected into U251 cells. RT-PCR was emplyed to detect the mRNA expression of Gli1 gene to select the siRNA interference fragment (siRNA-Gli1) that could most efficiently inhibit the mRNA expression of Gli1 gene. The mRNA and protein expressions of Gli1 gene at different times after siRNA-Gli1 transfection were detected to determine the time law of this interference. U251 cells at logarithmic phase were divided into 3 groups: siRNA-Gli1 group (transfection of selected siRNA-Gli1 fragments), siRNA-NC (transfection of siRNA fragments) and siRNA-N group (blank controls). The mRNA and protein expressions of Bcl-2, Bax 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 Gli1 mRNA expression was noted at U251-60 cells 48 h after the transfection therefore, locus 60 was the best interference fragment 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 mRNA and protein expressions of Bax gene were significantly up-regulated in the siRNA-Gli1 group as compared with those in the siRNA-N and siRNA-NC groups 48 h after transfection (P＜0.05). Silencing Gli1 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 G0 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 Gli1 gene can be effectively inhibited by specific siRNA targeting Gli1 gene in U251 cells and the proliferation of U251 cells can be significantly inhibited, which may possibly be related to that siRNA-Gli1 decreases the expressions of Bcl-2 and cycin D1 and alters the ratio of Bcl-2/Bax.
