新靶点CyclinE干扰RNA对人脑胶质瘤侵袭能力的影响

Effects of CyclinE RNA interference on the invasion ability of human brain glioma

目的构建人CyclinE新靶点的干扰RNA真核表达载体,观察CyclinE表达受抑制后的人脑胶质瘤细胞的侵袭能力的变化。方法构建4个新靶点CyclinE干扰RNA的真核表达载体后,用双酶切和碱基序列测定,转染载体到胶质瘤U251细胞株,将其分为U251组、PNC-U251组、PCyclinE-1-U251组、PCyclinE-2-U251组、PCyclinE-3-U251组、PCyclinE-4-U251组。通过RT-PCR的结果检测各组CyclinE mRNA的表达量,将干扰效果最好的一组用Transwell小室法检测侵袭能力的变化。结果成功构建了新靶点CyclinE干扰RNA真核表达载体即PCyclinE-1、PCyclinE-2、PCyclinE-3、PCyclinE-4,而且CyclinE mRNA表达受到抑制,其中PCyclinE-1-U251组的穿膜细胞数为(45.4±2.7)个,U251组和PNC-U251组的穿膜细胞数分别为(75.2±2.9)个和(74.4±3.5)个,U251组和PNC-U251组的穿膜细胞数比较,差异无统计学意义(t=1.23,P=0.31),PCyclinE-1-U251组和U251组的穿膜细胞数比较,差异有统计学意义(t=15.00,P=0.00),PCyclinE-1-U251组和PNC-U251组的穿膜细胞数比较,差异有统计学意义(t=13.81,P=0.00)。结论本实验构建了新靶点CyclinE干扰RNA真核表达载体,PCyclinE-1-U251细胞侵袭能力减弱。

Objective To construct the eukaryotic expression vector of RNA interference specific of CyclinE, and ob-serve the changes of invasion ability of U251 after the expression inhibition of CyclinE. Methods 4 new eukaryotic ex-pression vector of RNA interference specific of CyclinE were constructed, which were tested by restriction enzyme di-gestion and nucleotide sequence determined. Vectors were transfected by lipofectamine U251 cell lines. The expression levels of CyclinE mRNA were detected after transfection by RT-PCR. The change of cell invasion ability was detected with Transwell migration assay after CyclinE silence. Results Eukaryotic expression vector of New targets RNA inter-ference specific for CyclinE was constructed successfully, which were PCyclinE-1, PCyclinE-2, PCyclinE-3 and PCy-clinE-4, the result shows that the expression of CyclinE mRNA was inhibited, the number of transmembrane in the PCyclinE-1-U251 group was （45.4±2.7）, the number of U251 group and PNC-U251 group were （75.2±2.9）, （74.4±3.5）respectively, there was no statistically significant difference between U251 group and PNC-U251 group （t = 1.23,P=0.31）, and there was statistically significant difference between U251 group and PCyclinE-1-U251 group（t=15.00, P=0.00）, PCyclinE-1-U251 group and PNC-U251 group （t =13.81,P= 0.00）. Conclusion CyclinE RNA interfer-ence eukaryotic expression vector is successfully constructed, the invasion ability of interfering cells group is weak.