中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
8期
1759-1761
,共3页
胶质瘤%特异性核基质结合区结合蛋白-1%侵袭
膠質瘤%特異性覈基質結閤區結閤蛋白-1%侵襲
효질류%특이성핵기질결합구결합단백-1%침습
Glioma%Special AT-rich sequence binding protein 1%Invasion
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.
目的 探討特異性覈基質結閤區結閤蛋白-1(SATB1)短髮夾覈糖覈痠(shRNA)對人膠質瘤U251細胞侵襲的影響及其機製.方法 構建針對SATB1的shRNA重組質粒,採用電穿孔的方法轉染U251細胞中,分為對照組、空載體轉染組、重組質粒轉染組,採用逆轉錄-聚閤酶鏈反應(RT-PCR)檢測各組細胞SATB1 mRNA水平的變化,採用Western blot檢測各組細胞SATB1蛋白水平的變化,併對腫瘤細胞中相關功能蛋白的錶達進行分析;採用酶聯免疫吸附試驗法(ELISA)檢測細胞外基質金屬蛋白酶(MMP)-2和MMP-9濃度的變化;採用劃痕實驗和Transwell實驗評價腫瘤細胞侵襲能力的變化.結果 轉染後48 h,SATB1-shRNA重組質粒轉染組細胞外MMP-2的濃度為(69.4±3.5) μg/L,較對照組的(152.5±2.6)μg/L和空載體轉染組的(150.5±5.2)μg/L明顯降低,差異有統計學意義(P＜0.05).轉染後48 h,ELISA法檢測SATB1-shRNA重組質粒轉染組細胞外MMP-9的濃度為(40.6±2.4) μg/L,較對照組的(86.7±6.7)μg/L和空載體轉染組的(89.8±10.5)μg/L明顯降低,差異有統計學意義(P＜0.05).與對照組、空載體轉染組比較,重組質粒轉染組U251細胞SATB1、MMP-2和MMP-9的錶達下降,而基質金屬蛋白酶抑製因子2(TIMP-2)和SLC22A18錶達上調,差異有統計學意義(P＜0.05).劃痕實驗和Transwell實驗顯示重組質粒轉染組細胞侵襲能力明顯減弱.結論 針對SATB1的RNA榦擾可以明顯抑製U251膠質瘤細胞SATB1的錶達,對U251膠質瘤細胞的侵襲能力產生明顯抑製作用.
목적 탐토특이성핵기질결합구결합단백-1(SATB1)단발협핵당핵산(shRNA)대인효질류U251세포침습적영향급기궤제.방법 구건침대SATB1적shRNA중조질립,채용전천공적방법전염U251세포중,분위대조조、공재체전염조、중조질립전염조,채용역전록-취합매련반응(RT-PCR)검측각조세포SATB1 mRNA수평적변화,채용Western blot검측각조세포SATB1단백수평적변화,병대종류세포중상관공능단백적표체진행분석;채용매련면역흡부시험법(ELISA)검측세포외기질금속단백매(MMP)-2화MMP-9농도적변화;채용화흔실험화Transwell실험평개종류세포침습능력적변화.결과 전염후48 h,SATB1-shRNA중조질립전염조세포외MMP-2적농도위(69.4±3.5) μg/L,교대조조적(152.5±2.6)μg/L화공재체전염조적(150.5±5.2)μg/L명현강저,차이유통계학의의(P＜0.05).전염후48 h,ELISA법검측SATB1-shRNA중조질립전염조세포외MMP-9적농도위(40.6±2.4) μg/L,교대조조적(86.7±6.7)μg/L화공재체전염조적(89.8±10.5)μg/L명현강저,차이유통계학의의(P＜0.05).여대조조、공재체전염조비교,중조질립전염조U251세포SATB1、MMP-2화MMP-9적표체하강,이기질금속단백매억제인자2(TIMP-2)화SLC22A18표체상조,차이유통계학의의(P＜0.05).화흔실험화Transwell실험현시중조질립전염조세포침습능력명현감약.결론 침대SATB1적RNA간우가이명현억제U251효질류세포SATB1적표체,대U251효질류세포적침습능력산생명현억제작용.
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.
