微小RNA-22对髓母细胞瘤细胞恶性生物学行为的影响及其机制

Effect of microRNA-22 on malignant biological behavior of medulloblastoma cells and its mechanism

目的分析微小RNA-22(miR-22)对髓母细胞瘤细胞恶性生物学行为的影响,并探讨其可能作用机制。方法采用实时荧光定量PCR法检测正常人神经细胞RGC-5细胞和髓母细胞瘤细胞系UW402、UW473、DAOY和ONS-76细胞中miR-22的表达水平。将DAOY细胞分为对照组、miR-22 NC组、miR-22 mimics组、miR-22 mimics+740Y-P组,分别转染Lipofectamine^(TM) 2000试剂、miR-22 NC、miR-22 mimics、miR-22 mimics,同时,转染24 h后,miR-22 mimics+740Y-P组加入10μmol/L的740Y-P 200μL。采用CCK-8法和Transwell实验分别检测各组细胞的增殖和侵袭能力,采用流式细胞术检测细胞凋亡情况,蛋白免疫印迹法检测磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K、蛋白激酶B(Akt)、磷酸化Akt蛋白相对表达水平。将20只裸鼠随机分为对照组、miR-22 NC组、miR-22 mimics组、miR-22 mimics+740Y-P组,各5只,分别移植相应组别的细胞后,观察皮下移植瘤体积。结果与正常人神经细胞RGC-5细胞相比,miR-22在髓母细胞瘤细胞系中均呈低表达状态,且在DAOY细胞中的表达水平最低(均P<0.05)。与对照组和miR-22 NC组比较,miR-22 mimics组DAOY细胞的增殖和侵袭能力下降,凋亡率增高,PI3K和Akt磷酸化水平降低,且裸鼠的皮下移植瘤体积减小(均P<0.05);与miR-22 mimics组相比,miR-22 mimics+740Y-P组细胞的增殖和侵袭能力增强,凋亡率降低,PI3K和Akt磷酸化水平升高,且裸鼠的皮下移植瘤体积增大(均P<0.05)。结论上调miR-22表达可抑制髓母细胞瘤细胞恶性生物学行为,其机制可能与调控PI3K/Akt信号通路有关。

Objective To analyze the effect of microRNA-22(miR-22)on the malignant biological behavior of medulloblastoma cells,and to explore its possible mechanism of action.Methods Real-time fluorescent quantitative PCR was used to detect the expression levels of miR-22 in normal human neurocytes RGC-5 cells and medulloblastoma cell lines UW402,UW473,DAOY and ONS-76 cells.DAOY cells were divided into control group,miR-22 NC group,miR-22 mimics group and miR-22 mimics+740Y-P group which were transfected with Lipofectamine^(TM) 2000 kit,miR-22 NC,miR-22 mimics,and miR-22 mimics,respectively;in addition,after 24 hours of transfection,the miR-22 mimics+740Y-P group was added with 200μL of 10μmol/L 740Y-P.CCK-8 assay and Transwell experiment were used to detect the proliferation and invasion abilities of the cells in each group,respectively,flow cytometry to detect cell apoptosis,and Western blotting assay to detect the relative expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt)proteins.Twenty nude mice were randomly divided into control group(n=5),miR-22 NC group(n=5),miR-22 mimics group(n=5)and miR-22 mimics+740Y-P group(n=5)which were transplanted with corresponding cells.Subsequently,the volumes of subcutaneous transplanted tumors were observed.Results Compared with normal human neurocytes RGC-5 cells,the miR-22 was lowly expressed in medulloblastoma cell lines,and its lowest expression level was observed in DAOY cells(all P<0.05).Compared with the control and miR-22 NC groups,DAOY cells in the miR-22 mimics group exhibited declined proliferation and invasion abilities,an elevated apoptosis rate,reduced phosphorylation levels of PI3K and Akt,and decreased volume of subcutaneous transplanted tumors in nude mice(all P<0.05).Compared with the miR-22 mimics group,the miR-22 mimics+740Y-P group had increased proliferation and invasion abilities,a reduced apoptosis rate,higher phosphorylation levels of PI3K and Akt,and increased volume of subcutaneous transplanted tumors in nude mice(all P<0.05).Conclusion Up-regulation of miR-22 expression can inhibit the malignant biological behavior of medulloblastoma cells,and its mechanism may be related to the regulation of PI3K/Akt signaling pathway.