siRNA抑制PTTG1表达对人脑胶质瘤细胞增殖、侵袭性的影响

Effect of si RNA inhibition on PTTG1 expression on cell proliferation and invasion in human glioma

目的构建针对人脑胶质瘤细胞系的高效沉默PTTG1的RNAi载体;探讨PTTG1 siRNA干扰质粒对人脑胶质瘤细胞系U373增殖、侵袭性的影响。方法设计三对PTTG1基因干扰序列,合成能够特异干扰PTTG1的siRNA,然后将其与p Genesil2载体连接,构建pGenesil2-PTTG1 siRNA干扰载体,在脂质体作用下将PTTG1 siRNA质粒转染至胶质瘤U373细胞后,用RT-PCR和Western blot方法检测PTTG1 mRNA及蛋白表达水平的变化,通过细胞增殖实验、划痕实验和Transwell小室实验检测PTTG1对胶质瘤细胞增殖、迁移及侵袭能力的影响。结果在胶质瘤U373细胞转染3个不同的siRNA片段干扰PTTG1的表达,RTPCR检测结果显示,PTTG1 siRNA1干扰质粒对PTTG1表达的抑制作用最明显[(0.47±0.12)vs(1.00±0.15),P<0.01]。转染PTTG1 siRNA质粒的胶质瘤细胞U373在48h和72h细胞增殖能力均显著低于对照组。细胞划痕实验显示干扰PTTG1可明显缩短U373细胞的迁移距离;Transwell小室结果显示干扰PTTG1后U373的穿膜细胞百分率明显下降[(58.00±8.72)%vs(36.3±7.76)%,P<0.05]。结论 PTTG1 siRNA干扰质粒可明显抑制胶质瘤U373细胞PTTG1的表达,并可抑制胶质瘤细胞的增殖、侵袭及迁移能力。

Objective To construct RNAi vector of the efficient silencing PTTG1 of human glioma cell line and to explore the effect of PTTG1 siRNA interference plasmid on proliferation and invasion of human glioma cell line U373. Methods Three pairs of PTTG1 sequences were designed to synthesize siRNA which can interfere specifically with PTTG1. Then siRNA was connected with p Genesil2 vector to construct p Genesil2- PTTG1 siRNA interference vector. After transfection of PTTG1 siRNA plasmid to glioma U373 under the effect of liposome, PTTG1 mRNA and changes of protein level were detected by RT-PCR and Western blot method. Effect of PTTG1 on proliferation, migration and invasion of glioma cell was detected by cell proliferative assay, scratch adhesion test and Transwell experiment. Results Three different siRNA fragments interfered the expression of PTTG1 after transfection into the glioma cells U373. RT-PCR results showed that inhibition effect of PTTG1 siRNA1 interference plasmid on expression of PTTG1 was most significant [（0.47±0.12） vs（1.00±0.15）, P〈0.01]. Cell proliferation abilities of 48 h and 72 h of glioma cell U373 with transfection of PTTG1 siRNA were both significantly lower than the control group. Results of cell scratch adhesion test showed that interference with PTTG1 can obviously shorten the migration distance of U373 cell. Transwell experiment showed that the cell percentage of passed membrane cells of U373 after transfection of PTTG1 was significantly decreased [（58.00±8.72） % vs（36.3±7.76） %, P〈0.05]. Conclusion PTTG1 siRNA interference plasmid can significantly inhibit PTTG1 expression in U373 cell and abilities of cell proliferation, migration and invasion.