微小RNA-451对人脑U87胶质瘤细胞株增殖和侵袭影响机制的研究

Study on influence mechanism of miRNA-451 for the proliferation and invasion of U87 glioma cell lines in human brains

目的 探讨微小RNA-451（miRNA-451）通过靶标钙结合蛋白39（CAB39）调控缺氧诱导因子1 α（HIF-1α）的表达对人U87胶质瘤细胞株增殖和侵袭的影响.方法 培养人脑U87胶质瘤细胞,将其分为空白对照组、无义序列组及miRNA-451 mimics处理组（简称miRNA-451组）;将合成的寡核苷酸miRNA-451拟似物（miRNA-451 mimics）转染至U87胶质瘤细胞.实时定量PCR法检测转染后胶质瘤细胞miRNA-451的表达,Western blot法检测CAB39、肝激酶B1（LKBl）、HIF-lα蛋白的表达,应用流式细胞术检测细胞周期的变化,噻唑兰比色分析法（MTT）检测细胞增殖能力,Transwell 实验检测细胞侵袭能力的变化.结果 空白对照组、无义序列组、miRNA-451组的miRNA-451相对表达量分别为13.08 ±0.48、12.91±0.66、9.94 ±0.29,CAB39的相对表达量分别为2.85±0.05、2.40±0.04,0.41±0.02,LKB1的相对表达量为1.05±0.04、0.95±0.04、0.28±0.02,HIF-1α的相对表达量为0.60±0.02、0.54±0.03、0.14±0.05,差异均有统计学意义（P＜0.05）;细胞周期分析表明,空白对照组、无义序列组及miRNA-451组细胞G0/G1期DNA含量分别为60.48％、58.70％、75.58％,差异有统计学意义（P ＜0.05）;MTT法分析显示,miRNA-451组细胞生长明显受到抑制,第6天细胞增殖率仅为41.48％;Transwell法分析显示,miRNA-451组穿过Transwell小室的细胞数明显减少,仅为24个,低于空白对照组和无义序列组的51个和45个,差异均有统计学意义（P＜0.05）.结论 miRNA-451通过降低靶标CAB39的表达调控HIF-1α的表达,抑制人脑胶质瘤细胞的增殖和侵袭能力.

Objective To investigate the effects of microRNA-451 （miRNA-451） on the proliferation and invasion of U87 glioma cell lines in human brains by target calcium-binding protein 39 （CAB39） regulating the expression of hypoxia-inducible factor-1 a （HIF-1 α）.Methods The U87 glioma cells in human brains were cultured.They were divided into 3 groups：a blank control group,a nonsense sequence group,and a miRNA-451 treatment mimics group （briefly miRNA-451 group）.The synthetic oligonucleotide miRNA-451 analogs （miRNA-451 mimics） were transfected to U87 glioma cells.Real-time PCR was used to detect the expression of miRNA-451 after transfection of glioma cells.Western blot was used to detect the expression levels of CAB39,liver kinase B1 （LKB1）,and HIF-1α proteins.The flow cytometry was used to detect the changes of cell cycle.The methyl thiazolyl tetrazolium （MTT） colorimetric assay was used to detect cell proliferation ability.Transwell assay was used to detect the changes of cell invasion ability.Results The relative expression quantities of miRNA-451 in the blank control group,the nonsense sequence group,and the miRNA-451 group were 13.08 ±0.48,12.91 ±0.66,and 9.94 ±0.29,respectively;the relative expression quantities of CAB39 were 2.85 ± O.05,2.40 ± 0.04,and 0.41 ± 0.02,respectively;the relative expression quantities of LKB1 were 1.05 ±0.04,0.95 ±0.04,and 0.28 ±0.02,respectively;and the relative expression quantities of HIF-1α were 0.60 ±0.02,0.54 ±0.03,and 0.14 ±0.05,respectively.There were significant differences （P 〈 0.05）.The cell cycle analysis showed that the DNA contents of cell G0/G1 phase in the miRNA-451 group were 60.48%,58.70%,and 75.58%,respectively compared with the blank control and the nonsense sequence group.There were significant differences （P 〈 0.05）.The MTT assay showed that the cell growth was significantly inhibited in the miRNA-451 group and the cell proliferation rate was only 41.48% on the 6th day.The Transwell assay showed that the numbers of cells passing through Transwell chambers in the miRNA-451 group were decreased obviously,and.there were only 24.It was lower than 51 and 45 in the blank control group and the nonsense sequence group.There were significant differences （P 〈 0.05）.Conclusion miRNA-451 regulates the expression of HIF-1α by reducing the expression of target CAB39,and inhibits the proliferation and invasion ability of glioma cells in human brains.