转录因子Ascl1对人胶质瘤细胞增殖迁移的影响

Effects of Achaete-scute Homolog 1 on the Proliferation and Invasion of Human Glioma Cells

目的探讨转录因子Ascl1(Achaete-scute homolog 1)对人胶质瘤细胞U87增殖和侵袭的影响,初步探索其调控机制。方法将Ascl1慢病毒感染U87细胞;通过观察绿色荧光表达、Western blot实验和q-PCR实验鉴定U87细胞中Ascl1的表达;用CCK-8实验检测Ascl1对U87细胞增殖能力的影响;Transwell侵袭实验检测Ascl1对U87细胞侵袭能力的影响;流式细胞术检测Ascl1对U87细胞周期的调控,并通过Western blot检测各组细胞内细胞周期相关蛋白c-myc、Cyclin D1、CDK4和P27的表达变化。结果 U87细胞感染慢病毒后高表达Ascl1,效率可达90%以上;高表达Ascl1的U87细胞在450nm处光密度(OD)值下降;高表达Ascl1的U87细胞穿过小室基底膜的细胞数减少;Ascl1可以使U87细胞的细胞周期受阻于G0/G1期,G1期向S期转换受到抑制;Ascl1下调细胞周期相关蛋白c-myc、Cyclin D1和CDK4的表达,上调P27的表达。结论 Ascl1能够减弱人胶质瘤细胞U87的增殖和侵袭能力,该抑制作用可能与细胞周期的调控有关。

Objective To investigate the effect of Achaete-scute homolog 1（Ascl1）on the proliferation and invasion of human glioma cells（U87 cells）and to explore the regulation mechanism. Methods The U87 cells were infected by Ascl1 lentiviruses. The overexpression of Ascl1 in U87 cells were identified by observing the green fluorescence,Western blot and q-PCR assays. CCK-8 was used to determine the effect of Ascl1 on the proliferation of U87 cells. The effect of Ascl1 on the invasion of U87 cells was checked by transwell invasion assay. The cell cycle of U87 cells regulated by Ascl1 was investigated by flow cytometry,and Western blot was taken to detect the expression of c-myc,Cyclin D1,CDK4 and P27 in U87 cells. Results It was determined that Ascl1 was overexpressed in U87 cells after infection of lentivirus. The infection efficiency exceeded 90 percent. Compared with the control group cells,CCK-8 results showed that the optical density（OD）at 450 nm decreased in Ascl1 overexpression U87 cells. Transwell invasion assay showed that the number of cells which passed through the membrane was reduced in Ascl1 overexpression U87 cells. Flow cytometry demonstrated that the cell cycle of Ascl1 overexpression U87 cells was arrested in G0/G1 phase,and the conversion of G1 to S phase was inhibited. At the same time,Ascl1 down-regulated the expression of c-myc,Cyclin D1 and CDK4,and up-regulated the expression of P27. Conclusion Ascl1 can reduce the proliferation and invasion of U87 cells,which is related to the regulation of cell cycle.