ZBTB3表达对胶质母细胞瘤细胞增殖和克隆形成的调控

Regulation of ZBTB3 expression on proliferation and clone formation of glioblastoma cells

目的:检查人胶质母细胞瘤(glioblastoma,GBM)组织和细胞系中ZBTB3的表达,并探讨ZBTB3对GBM细胞增殖和克隆形成的影响及其调控机制。方法:通过GEPIA2数据库分析GBM患者肿瘤组织中ZBTB3的表达情况。RT-PCR、qPCR和Western blot检测GBM细胞系(U251、U373、U87)中ZBTB3的mRNA和蛋白表达水平,筛选出ZBTB3表达最高的U87细胞。CCK-8和克隆形成实验检测沉默ZBTB3基因对U87细胞增殖和克隆形成的影响。用p38MAPK、AMPK、Akt1抑制剂处理U87细胞后,Western blot检测p38MAPK、AMPK、Akt1的磷酸化水平,RT-PCR、qPCR和Western blot测定ZBTB3的mRNA和蛋白表达水平,CCK-8和克隆形成实验检测细胞增殖和克隆形成。结果:GBM患者肿瘤组织中ZBTB3的表达显著高于正常组织。U251、U373和U87细胞中均可见ZBTB3的表达,其中U87细胞表达最高。沉默ZBTB3基因能明显抑制U87细胞的增殖和克隆形成。抑制AMPK既能显著降低U87细胞ZBTB3的表达水平,又可明显减弱U87细胞增殖和克隆形成。结论:GBM组织和细胞系中ZBTB3的表达显著上调,GBM细胞中AMPK活化并上调ZBTB3基因的表达,促进GBM细胞的增殖和克隆形成。

Objective:This study aims to examine the expression of ZBTB3 in human glioblastoma(GBM)tissues and cell lines,and to explore the effects of ZBTB3 on the proliferation and clonal formation of GBM cells and their regulatory mechanism.Methods:The expression of ZBTB3 in tumor tissues of GBM patients was analyzed by GEPIA2 database.The mRNA and protein expression levels of ZBTB3 in GBM cell lines(U251,U373,U87)were detected by RT-PCR,qPCR and Western blot,and U87 cell line was identified with the highest expression of ZBTB3.CCK-8 and clonal formation assay were used to examine the effects of silencing ZBTB3 on the proliferation and clonal formation of U87 cells.U87 cells were treated with p38MAPK,AMPK and Akt1 inhibitors,and the phosphorylation levels of p38MAPK,AMPK and Akt1 were detected by Western blot.ZBTB3 mRNA and protein levels were detected by RT-PCR,qPCR and Western blot.Cell proliferation and clone formation were examined by CCK-8 and clone formation assay.Results:The expression of ZBTB3 in tumor tissues of GBM patients was significantly higher than that in normal tissues.The expression levels of ZBTB3 in U251,U373 and U87 cell lines were examined,and the highest expression in U87 cells was observed.Silencing ZBTB3 markedly inhibited the proliferation and clonal formation of U87 cells.AMPK inhibition could not only obviously reduce the expression level of ZBTB3 in U87 cells,but also markedly attenuate the proliferation and clonal formation of U87 cells.Conclusion:The expression of ZBTB3 is obviously increased in GBM tissues and cells,and the AMPK-up-regulated ZBTB3 expression promotes the proliferation and clonal formation of GBM cells.