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前期的研究证明,人腺苷酸活化蛋白激酶5(ARK5)通过Gab2-Akt-ARK5通路,促进胶质瘤的侵袭。然而,ARK5的调节机制尚不清楚。本研究旨在探讨miR-424是否通过与ARK5 mRNA结合,进而影响胶质瘤侵袭。通过Targetscan找到与ARK5的3’-UTR区互补结合的microRNA(miR-424)。运用实时荧光定量PCR(qRT-PCR)检测不同胶质瘤细胞系中miR-424表达水平,发现3种胶质瘤母细胞(高级别胶质瘤细胞)株中,miR-424表达量均不同程度低于低级别胶质瘤H4细胞株。miR-424及miR-424抑制剂质粒转染胶质瘤细胞系H4、LN-229并结合蛋白质印迹法显示,miR-424可负向调控ARK5蛋白表达。qRT-PCR显示,在胶质瘤细胞内过表达miR-424后,ARK5mRNA无显著变化,提示miR-424在翻译水平影响ARK5蛋白表达。Transwell侵袭实验显示,过表达miR-424导致胶质瘤细胞体外侵袭能力明显减弱。双荧光素酶基因报告检测显示,miR-424能与ARK5 mRNA的3’-UTR结合,抑制荧光素酶活性。上述结果提示,miR-424可以结合ARK5mRNA的3’-UTR而抑制ARK5蛋白翻译,从而抑制胶质瘤细胞侵袭。
Preliminary studies have shown that human adenylate activated protein kinase 5 (ARK5) promotes glioma invasion through the Gab2-Akt-ARK5 pathway. However, the regulatory mechanism of ARK5 is not clear. This study aimed to investigate whether miR-424 binds to ARK5 mRNA and thereby affects glioma invasion. Targetscan find microRNAs (miR-424) that complementarily bind to the 3’-UTR region of ARK5. The expression of miR-424 in different glioma cell lines was detected by real-time fluorescence quantitative PCR (qRT-PCR), and the expression levels of miR-424 in the three glioma blast cells (high grade glioma) were different Less than low-grade glioma H4 cell lines. miR-424 and miR-424 inhibitor plasmids were transfected into glioma cell lines H4 and LN-229 and combined with Western blotting, miR-424 negatively regulated ARK5 protein expression. qRT-PCR showed no significant changes in ARK5 mRNA after overexpression of miR-424 in glioma cells, suggesting that miR-424 may affect ARK5 protein expression at translational level. Transwell invasion assay showed that overexpression of miR-424 resulted in a significant decrease in in vitro invasiveness of glioma cells. Dual luciferase reporter gene assay showed that miR-424 can bind to the 3’-UTR of ARK5 mRNA and inhibit luciferase activity. The above results suggest that miR-424 can inhibit ARK5 protein translation by binding to the 3’-UTR of ARK5 mRNA, thereby inhibiting the invasion of glioma cells.