中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
7期
1472-1474
,共3页
楚胜华%冯东福%马延斌%朱志安
楚勝華%馮東福%馬延斌%硃誌安
초성화%풍동복%마연빈%주지안
胶质瘤%异性核基质结合区结合蛋白-1%细胞增殖
膠質瘤%異性覈基質結閤區結閤蛋白-1%細胞增殖
효질류%이성핵기질결합구결합단백-1%세포증식
Glioma%Special AT-rich sequence binding protein 1%Cell proliferation
目的 探讨异性核基质结合区结合蛋白-1(SATB1)短发夹核糖核酸(shRNA)对高表达SATB1的人胶质瘤细胞株U251和SHG44增殖的影响及其机制.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Western blot检测各组细胞SATB1基因和蛋白的表达,以噻唑蓝(MTT)法测各组细胞的增殖活性,Western blot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(FoxO1)、细胞周期蛋白(Cyclin D1)、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SATB1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SATB1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FoxO1水平降低,Cyclin D1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SATB1的RNA干扰可以明显抑制转染人胶质瘤细胞株U251和SHG44细胞SATB1的表达和细胞增殖,可能与抑制AKT/FoxO1信号通路,调控下游基因Cyclin D1、p27蛋白的表达有关.
目的 探討異性覈基質結閤區結閤蛋白-1(SATB1)短髮夾覈糖覈痠(shRNA)對高錶達SATB1的人膠質瘤細胞株U251和SHG44增殖的影響及其機製.方法 構建針對SATB1的shRNA重組質粒,採用電穿孔的方法轉染至U251和SHG44細胞中,分為對照組、空載體轉染組、重組質粒轉染組,分彆通過逆轉錄-聚閤酶鏈反應(RT-PCR)和Western blot檢測各組細胞SATB1基因和蛋白的錶達,以噻唑藍(MTT)法測各組細胞的增殖活性,Western blot檢測各組細胞的蛋白激酶B(AKT)、燐痠化AKT、燐痠化扠頭轉錄因子(FoxO1)、細胞週期蛋白(Cyclin D1)、p27蛋白的錶達.結果 與對照組(1.22±0.06和1.24 ±0.07)、空載體轉染組(1.20±0.05和1.29±0.07)比較,重組質粒轉染組(0.24±0.03和0.27±0.02)U251和SHG44細胞SATB1基因的錶達下降；與對照組(1.02±0.06和1.03±0.09)、空載體轉染組(0.96±0.06和0.98±0.08)比較,重組質粒轉染組(0.19±0.02和0.21±0.03) U251和SHG44細胞SATB1蛋白的錶達和細胞增殖能力均下降,細胞燐痠化AKT和燐痠化FoxO1水平降低,Cyclin D1蛋白錶達減少,p27蛋白錶達增多,差異均有統計學意義(P＜0.05).結論 針對SATB1的RNA榦擾可以明顯抑製轉染人膠質瘤細胞株U251和SHG44細胞SATB1的錶達和細胞增殖,可能與抑製AKT/FoxO1信號通路,調控下遊基因Cyclin D1、p27蛋白的錶達有關.
목적 탐토이성핵기질결합구결합단백-1(SATB1)단발협핵당핵산(shRNA)대고표체SATB1적인효질류세포주U251화SHG44증식적영향급기궤제.방법 구건침대SATB1적shRNA중조질립,채용전천공적방법전염지U251화SHG44세포중,분위대조조、공재체전염조、중조질립전염조,분별통과역전록-취합매련반응(RT-PCR)화Western blot검측각조세포SATB1기인화단백적표체,이새서람(MTT)법측각조세포적증식활성,Western blot검측각조세포적단백격매B(AKT)、린산화AKT、린산화차두전록인자(FoxO1)、세포주기단백(Cyclin D1)、p27단백적표체.결과 여대조조(1.22±0.06화1.24 ±0.07)、공재체전염조(1.20±0.05화1.29±0.07)비교,중조질립전염조(0.24±0.03화0.27±0.02)U251화SHG44세포SATB1기인적표체하강；여대조조(1.02±0.06화1.03±0.09)、공재체전염조(0.96±0.06화0.98±0.08)비교,중조질립전염조(0.19±0.02화0.21±0.03) U251화SHG44세포SATB1단백적표체화세포증식능력균하강,세포린산화AKT화린산화FoxO1수평강저,Cyclin D1단백표체감소,p27단백표체증다,차이균유통계학의의(P＜0.05).결론 침대SATB1적RNA간우가이명현억제전염인효질류세포주U251화SHG44세포SATB1적표체화세포증식,가능여억제AKT/FoxO1신호통로,조공하유기인Cyclin D1、p27단백적표체유관.
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.
