沉默特异性核基质结合区结合蛋白-1对胶质瘤U251细胞侵袭的影响

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

目的 探讨特异性核基质结合区结合蛋白-1（SATB1）短发夹核糖核酸（shRNA）对人胶质瘤U251细胞侵袭的影响及其机制.方法 构建针对SATB1的shRNA重组质粒,采用电穿孔的方法转染U251细胞中,分为对照组、空载体转染组、重组质粒转染组,采用逆转录-聚合酶链反应（RT-PCR）检测各组细胞SATB1 mRNA水平的变化,采用Western blot检测各组细胞SATB1蛋白水平的变化,并对肿瘤细胞中相关功能蛋白的表达进行分析;采用酶联免疫吸附试验法（ELISA）检测细胞外基质金属蛋白酶（MMP）-2和MMP-9浓度的变化;采用划痕实验和Transwell实验评价肿瘤细胞侵袭能力的变化.结果 转染后48 h,SATB1-shRNA重组质粒转染组细胞外MMP-2的浓度为（69.4±3.5） μg/L,较对照组的（152.5±2.6）μg/L和空载体转染组的（150.5±5.2）μg/L明显降低,差异有统计学意义（P＜0.05）.转染后48 h,ELISA法检测SATB1-shRNA重组质粒转染组细胞外MMP-9的浓度为（40.6±2.4） μg/L,较对照组的（86.7±6.7）μg/L和空载体转染组的（89.8±10.5）μg/L明显降低,差异有统计学意义（P＜0.05）.与对照组、空载体转染组比较,重组质粒转染组U251细胞SATB1、MMP-2和MMP-9的表达下降,而基质金属蛋白酶抑制因子2（TIMP-2）和SLC22A18表达上调,差异有统计学意义（P＜0.05）.划痕实验和Transwell实验显示重组质粒转染组细胞侵袭能力明显减弱.结论 针对SATB1的RNA干扰可以明显抑制U251胶质瘤细胞SATB1的表达,对U251胶质瘤细胞的侵袭能力产生明显抑制作用.

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.