反义及显性负调节AKT2抑制胶质瘤侵袭和迁移的实验研究

Study on the Inhibitory Effect of Anti-sense and Dominant-negative AKT2 Constructs on Glioma Cell Invasion and Migration

近来的研究显示AKT激酶家族成员在调节细胞生长、增殖、存活和代谢等方面的信号传导系统中起着极为重要的作用。本文以AKT2为对象,研究反义AKT2 cDNA(antisense AKT2,AS-AKT2)和显性负调节AKT2(dominant negative AKT2,DN-AKT2)cDNA构建体调控恶性胶质瘤侵袭性生长的作用及其机制。方法:利用反义AKT2 cDNA和显性负调节AKT2cDNA构建体转染鼠脑胶质瘤细胞系C6,分别在RNA水平和蛋白水平阻断AKT2通路,将野生型和转染pLXSN空载体的C6细胞作为对照。原位杂交和蛋白印迹鉴定后,Transwell和球体培养法分析侵袭能力,球体培养和划痕实验研究细胞迁移能力,明胶酶谱分析转染前后MMP2/9酶活性变化。结果:原位杂交和蛋白印迹结果表明,C6胶质瘤细胞系在mRNA和蛋白水平均高表达AKT2;转染AS-AKT2后AKT2表达被显著抑制,但转染DN-AKT2后AKT2表达无明显变化。C6细胞转染AKT2构建体后应用体外侵袭和迁移的实验量化分析这种效应后发现:利用Matrigel夹心法和Transwell方法研究发现,C6细胞转染AKT2构建体后侵袭能力下降46%～57%,细胞迁移能力下降23%～42%。明胶酶谱分析发现MMP2/9和pro-MMP9的活性被明显抑制。结论:AKT2可以通过调节MMP2/9活性调控恶性胶质瘤侵袭性生长,因此AKT2可能成为抑制胶质瘤侵袭性生长的新靶标。

BACKGROUND & OBJECTIVE: According to recently studies, the AKT kinase family mem-bers have emerged as central roles in the signaling cascades that regulate cell growth, proliferation, survival and vari-ous aspects of intermediary metabolism. In this paper, we chose the AKT2 to investigate the inhibitory effect of anti-sense (AS-AKT2) and dominant negative (DN-AKT2) serine/threonine kinase 2 (AKT2) constructs on invasion and migration of glioma cells and its possible mechanism. METHODS: Rat C6 glioblastoma cells were transfected with AS-AKT2 and DN-AKT2 constructs to block AKT2 expression in mRNA and protein level.Parental C6 cells and C6 cells transfected with empty vector pLXSN were used as controls. The expression of AKT2 in all cell lines were indentified by in situ hybridization and Western blot analysis.Tumor invasion was examined by Transwell and spheroid-Matrigel method, and cell migra-tion by spheroid and wound-healing method.The enzyme activities of the extracellular ma- trix degradation in MMP2/9 were evaluated by using gelatin zymography. RESULTS: Over-expression of AKT2 were identified by in situ hybridization and Western blot analysis in parental C6 cells and pLXSN-transfected cells. AKT2 expression was inhibited in AS - AKT2 transfected cells. However, there was no significant change in AKT2 expression in DN-transfected cells. Compared with that of C6 cells and LXSN transfected cells, tumor inva-sion of As-AKT2 transfected cells decreased 46%-57% by Transwell and spheroid -Matrigel method, and tu-mor cell migration dropped down 23%-42% by spheroid and wound-healing method, MMP2/9 activities re-duced 31%-38% by gelatin zymography. The inhibitory effects on cell invasion and migration showed no statistical-ly difference between AS-AKT2 transfected cells and DN-AKT2 transfected cells. CONCLUSIONS: AKT2 may exert pivotal role in invasion in glioma cells through regulation of MMP2/9 activities, so AKT2 may be an option-al candidate for gene therapy of gliomas in invasion.