质粒介导外源性微小RNA干扰PTTG1表达抑制恶性人脑胶质瘤细胞增殖和侵袭

Introduction of plasmid-mediated exogenous microRNA to silence PTTGI gene expression and inhibit proliferation and invasiveness of glioma cells

目的观察垂体瘤转化基因1（PTTG1）在恶性人脑胶质瘤U251细胞增殖和侵袭中的作用。方法设计并合成2对靶向PTTG1 mRNA的寡核苷酸片段．将其连接pcDNA6．2-GW／EmGFP-miR载体构建表达微小RNA真核载体MIR-1、MIR-2；将连接产物转化到大肠杆菌DH5d感受态细胞，经PCR筛选阳性克隆、测序鉴定；脂质体转染法将重组质粒转入U251细胞，同时设转染阴性对照序列的阴性对照组（转染Neg组）和空白组：实时荧光定量聚合酶链式反应（real-time PCR）分析肿G1 mRNA抑制效率：Western印迹测定PTTG1蛋白的表达量改变；MTT法检测U251细胞增殖的改变：Matrigel侵袭实验检测U251细胞侵袭力的改变。结果转染后，转染MIR-2组PTTG1 mRNA较空白组和转染Neg组下降为87、6％．PTTG1蛋白表达明显少于其他组：转染MIR-2组U251细胞不同时间点生长抑制率为10．7％～34．7％：Matrigel侵袭实验结果示MIR-2组穿过人工基底膜U251细胞数减少．相对百分率为12.3％±1．0％，明显低于空白组（24．7％±1．4％）和转染Neg组（24．0％±2．0％）．差异有统计学意义（P〈0．05）。结论人脑胶质瘤U251细胞的PTTG1表达可被质粒导入的外源性微小RNA有效抑制．进而抑制细胞增殖．逆转恶性胶质瘤U251细胞侵袭、浸润。

Objective To investigate the role of pituitary tumor transforming gene 1 （PTTG1） in the growth and invasion of human glioma cell line by introduction of exogenous microRNA to silence PTTGI gene expression. Methods Two double-stranded DNA pcDNA6.2-GW/EmGFP-miR vectors （MIR- 1, MIR-2） targeting human PTTG 1 mRNA and a negative control plasmid （Neg） were constructed, and were transfected into human U251 cells with high metastatic potentials. Real-time PCR and Western blotting were used to quantify the mRNA and protein levels of PTTG1, respectively. Proliferation and invasiveness of transfected U251 cells were analyzed by MTT assay and Matrigel invasion assay. Results After transfection, Expression of PTTGI mRNA was inhibited significantly with inhibitory rates of 87.6% in MIR-2 group, and the protein levels were significantly lower than those of the other groups. There was significant difference in cellular growth rate among the 3 groups. The growth inhibiting rates in the MIR-2 group are 10.7%-34.7%. The migrating number of U251 cells transfected with MIR-2 with relative percentage （12.3±1.0）% was also significantly decreased as compared the Neg group （24.7±1.4）% and Mock group （24.0±2.0）%. Conclusion Introduction of exogenous miRNA to U251 cell line by transfection of MIR-2 can effectively reduce the PTTG1 expression, which can significantly inhibit the proliferation and decrease the invasiveness of glioma cells.