微小RNA-200a对人脑胶质瘤细胞恶性生物学行为的影响

Researches of microRNA - 200a on malignant biological behavior of human glioma cells

目的探讨微小RNA（miRNA，miR）-200a对胶质瘤细胞U251增殖、凋亡、迁移、细胞周期的影响及作用机制。方法利用脂质体瞬时转染法将miR-200a模拟物（miR-200a组）和无义序列（Mock组）转染至体外常规培养人脑胶质瘤细胞株U251，同时设立Blank组（Blank组）。转染48h后实时定量反转录聚合酶链反应（RT-qPCR）检测3组细胞miR-200a的表达；细胞计数盒8（CCK-8）法检测检测3组细胞增殖能力；划痕实验检测各组细胞迁移能力；流式细胞术检测细胞凋亡和细胞周期，Westernblot检测各组细胞锌指E．盒结合同源异形盒1（ZEB1）和细胞周期蛋白D1（CyelinD1）蛋白的表达。结果转染后48h，与Mock组和Blank组比较，miR-200a组细胞中miR-200a相对表达量增高（分别为17．51±0．89，1．46±0．33，1．66±0．13），差异有统计学意义（P=0．000、0．000）；miR-200a组细胞在转染12h后增殖率开始降低，差异有统计学意义（P=0．028、0．010）；miR-200a组细胞凋亡率增加（P=0．001、0．001），G0／G1期细胞比例增加（P=0．011、0．013），S期细胞比例明显降低（P=0．024、0．008）；miR-200a组细胞迁移能力明显下降（P=0．031、0．030），Western blotting结果显示调控上皮-间充质转化（EMT）的主要分子ZEBl（P=0．042、0．034）及调控细胞周期进程的分子CyclinD1表达均明显下调，差异有统计学意义（P=0．020、0．013）。结论上调miR-200a的表达可以促进胶质瘤细胞凋亡，阻滞其细胞周期进程，抑制其迁移能力。

Objective To investigate the effect of microRNA （ miRNA, miR） - 200a on proliferation, apoptosis, migration and cell cycle of glioma cell line U251 and its possible mechanism. Methods miR- 200a mimics （miR-200a group） and nonsense sequences （Mock group） were transfected into human glioma ceil line U251 in vitro by liposome transient transfection. Blank group （ Blank group） group）. The expression of miR - 200a was detected by real - time quantitative reverse transcriptase - polymerase chain reaction （ RT - qPCR） after transfeetion for 48 hours. The cell proliferation ability of the three groups was detected by cell counting kit - 8 （ CCK - 8 ） method. The cell migration ability was detected by scratches. Cell apoptosis and cell cycle were detected by flow cytometry The expression of ZEB1 and Cyelin D1 protein was detected by Western blotting. Results Compared with the Mock and Blank groups, the relative expression of miR -200a in miR -200a ceils was significantly higher than that in the untreated group （ P = 0. 000, 0. 000 ）. The cells of miR - 200a cells were significantly different （ P = 0. 028, 0. 010 ）. The cell migration ability of miR -200a group was significantly decreased （P = 0. 031,0. 030 ）, the proportion of G0/Gj phase cells in miR - 200a group was increased （ P = 0.011, 0. 013 ）, the proportion of S phase cells was significantly decreased （ P = 0. 024,0.008 ） , and the apoptosis rate of miR - 200a group was sig- nificantly lower than that of miR - 200a group （ P =0.001,0.001 ）. Western blotting showed that the ex- pression of ZEB1 （P = 0. 042, 0. 034） and the expression of Cyclin D1 were significantly down -regulated （ P = 0. 020, 0. 013 ）. Conclusion Upregnlation of miR - 200a can promote glioma cell apoptosis, block its cell cycle progression and inhibit its migration ability, which is expected to be a new target for the treat- ment of glioma.