Influence of special AT-rich sequence binding protein 1 silencing on the invasion of glioma U251 cells

Abstract

Objective To study the effect of special AT-rich sequence binding protein 1 (SATB1) short hairpin RNA (shRNA) on the invasion of human glioma cells,and explore its mechanism.Methods The recombinant plasmid of small hairpin RNA targeting SATB1 gene was constructed,and transfected into glioma U251 cells by electroporation.Untransfected group,control-shRNA-green fluorescent protein (GFP) group and SATB1-shRNA group were set up in the experiment.The expression of SATB1 mRNA and protein in U251 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively.The expression of some functional proteins was also studied by Western blotting.Enzyme-linked immunosorbent assay (ELISA) was used to examine the changes of concentration of ectocytic matrix metalloproteinases 2 (MMP-2) and MMP-9.The invasion ability of U251 cells in the 3 groups was evaluated by scarification and Transwell assays.Results At 48 h after transfection,the concentration of extracellular MMP-2 in the SATB1-shRNA group [(69.4 ± 3.5) μg/L] was significantly lower than in the control group [(152.5 ± 2.6) μg/L] and the control-shRNA-GFP group [(150.5 ± 5.2) μg/L] (P ＜ 0.05 for both).The concentration of extracellular MMP-2 in the SATB1-shRNA group [(40.6 ±2.4) μμg/L] was significantly lower than in the control group [(86.7 ± 6.7) μg/L] and the control-shRNA-GFP group [(89.8 ± 10.5) μg/L] (P ＜ 0.05).As compared with the untransfected group and control-shRNA-GFP group,the SATB1-shRNA group showed significantly lower expression level of MMP-2 and MMP-9 (P ＜ 0.05).Meanwhile tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and SLC22A18 in the SATB1-shRNA group was significantly up-regulated.ELISA also indicated obvious changes of concentration of ectocytic MMP-2 and MMP-9.The scarification and Transwell assays revealed that the invasion ability in the SATB1-shRNA group was significantly decreased as compared with that in the rest two groups (P ＜ 0.05).Conclusion SATB1-targeted RNA intereference could inhibit the expressions of SATB1 and decrease the invasion ability of U251 cells in vitro. Key words: Glioma; Special AT-rich sequence binding protein 1 ; Invasion