RNA干扰抑制异性核基质结合区结合蛋白-1表达对人胶质瘤细胞增殖的影响

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

目的探讨异性核基质结合区结合蛋白．1（SATBl）短发夹核糖核酸（shRNA）对高表达SATBl的人胶质瘤细胞株U251和SHCcM增殖的影响及其机制。方法构建针对SATBl的shRNA重组质粒，采用电穿孔的方法转染至U251和sHG44细胞中，分为对照组、空载体转染组、重组质粒转染组，分别通过逆转录-聚合酶链反应（RT—PCR）和Westernblot检测各组细胞SATBI基因和蛋白的表达，以噻唑蓝（M33＂）法测各组细胞的增殖活性，Westernblot检测各组细胞的蛋白激酶B（AKT）、磷酸化AKT、磷酸化叉头转录因子（Fox01）、细胞周期蛋白（CyclinD1）、p27蛋白的表达。结果与对照组（1．22±0．06和1．24±0．07）、空载体转染组（1．20±0．05和1．29±0．07）比较，重组质粒转染组（0．24±0．03和0．27±0．02）U251和SHG44细胞SATBl基因的表达下降；与对照组（1．02±0．06和1．03±0．09）、空载体转染组（0．96±0．06和0．98±0．08）比较，重组质粒转染组（0．19±0．02和0．21±0．03）U251和SHG44细胞SATBl蛋白的表达和细胞增殖能力均下降，细胞磷酸化AKT和磷酸化Fox01水平降低，CyclinD1蛋白表达减少，p27蛋白表达增多，差异均有统计学意义（P〈0．05）。结论针对SATBl的RNA干扰可以明显抑制转染人胶质瘤细胞株U251和SHG44细胞SATBl的表达和细胞增殖，可能与抑制AKT／FoxOI信号通路，调控下游基因CyclinD1、p27蛋白的表达有关。

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 （RT- PCR） and Western blotting respectively. The viability of cells was determined by using methyl thiazol tetrazo- lium （MTF） assay. The effects of SATB1 shRNA on protein kinase B （AKT）/forhead box protein O1 （ FoxOl ） pathway and protein expression of Cyclin D1 and p27 were studied by using Western blotting. Re- suits The expression levels of SATB1 mRNA in U251 and SHG44 ceils were detected in a significantly low- er proportion in SATBI-shRNA group （0. 24 ±0. 03 and O. 27 ±0. 02） than in untransfected group （ 1.22 ± 0.06 and 1.24 ±0.07） and control-shRNh-GFP group （1.20 ±0. 05 and 1.29 ±0. 07）. The expression lev- els 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 O. 98 ± 0. 08 ）. The proliferation rate in SATBI-shRNA group was significantly decreased as compared with that untransfected and control-shRNA-GFP groups. Lev- els of phosphorylated AKT and phosphorylated FoxO1 were significantly decreased in SATBI-shRNA group as compared with untransfected and control-shRNA-GFP groups. Moreover, Cyclin D1 expression was de- creased, while p27 protein expression was up-regulated in SATBI-shRNA group as compared with untrans- fected and control-shRNA-GFP groups. Conclusion SATBl-targeted RNA interference could inhibit the ex- pression 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.