Effects of special AT-rich sequence binding protein 1 silencing upon the proliferation of human glioma cells by RNA interference

Abstract

Objective To investigate the effect of special AT-rich sequence binding protein 1 (SATB1) short hairpin RNA (shRNA) on the proliferation of human glioma U251 and SHG44 cells,and explore its mechanism.Methods The recombinant plasmid of small hairpin RNA targeting SATB1 gene was constructed,and transfected into glioma U251 and SHG44 cells by electroporation.The expression of SATB1 mRNA and protein in the cells was detected by using reverse transcriptase-polymerase chain reaction (RTPCR) and Western blotting respectively.The viability of cells was determined by using methyl thiazol tetrazolium (MTT) assay.The effects of SATB1 shRNA on protein kinase B (AKT)/forhead box protein O1 (FoxO1) pathway and protein expression of Cyclin D1 and p27 were studied by using Western blotting.Results The expression levels of SATB1 mRNA in U251 and SHG44 cells were detected in a significantly lower proportion in SATB1-shRNA group (0.24 ±0.03 and 0.27 ±0.02) than in untransfected group (1.22 ±0.06 and 1.24 ± 0.07) and control-shRNA-GFP group (1.20 ± 0.05 and 1.29 ± 0.07).The expression levels of SATB1 protein in U251 and SHG44 cells were detected in a significantly lower proportion in SATB1-shRNA group (0.19 ±0.02 and 0.21 ±0.03) than in untransfected group (1.02 ±0.06 and 1.03 ±0.09)and control-shRNA-GFP group (0.96 ± 0.06 and 0.98 ± 0.08).The proliferation rate in SATB1-shRNA group was significantly decreased as compared with that untransfected and control-shRNA-GFP groups.Levels of phosphorylated AKT and phosphorylated FoxO1 were significantly decreased in SATB1-shRNA group as compared with untransfected and control-shRNA-GFP groups.Moreover,Cyclin D1 expression was decreased,while p27 protein expression was up-regulated in SATB1-shRNA group as compared with untransfected and control-shRNA-GFP groups.Conclusion SATB1-targeted RNA interference could inhibit the expression of SATB1 and the cell proferation,which might be related to the suppression of AKT/FoxO1 pathway and the regulation of expression of their downstream molecules such as Cyclin D1 and p27. Key words: Glioma; Special AT-rich sequence binding protein 1; Cell proliferation