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目的利用RNA干扰(RNAi)在人胶质瘤U251细胞中沉默CDKL1(cyclin-dependent kinase-like 1)基因的表达,探讨其对肿瘤细胞增殖和凋亡的影响。方法构建针对CDKL1基因的短发卡(shRNA)慢病毒表达载体,包装成病毒颗粒并感染人胶质瘤U251细胞,采用实时定量聚合酶链反应和Western印迹法验证CDKL1基因的mRNA和蛋白的表达,确定其抑制效率。采用甲基噻唑基四唑(MTT)比色法和应用Annexin V染色流式细胞仪检测细胞增殖和凋亡情况,观察沉默CDKL1基因对U251细胞增殖与凋亡表型的影响。结果 CDKL1-shRNA能有效抑制U251细胞中CDKL1基因的mRNA和蛋白的表达。CDKL1基因沉默后U251各时相的光密度值较感染无义序列的细胞均有降低趋势,第5天时差异有统计学意义(P<0.05)。同时,抑制CDKL1的U251细胞凋亡比例从无义小干扰RNA(siRNA)序列对照组的(9.80±0.21)%上升至(19.26±0.33)%,差异有统计学意义(P<0.05)。结论采用RNAi方法能有效沉默CDKL1基因的表达,同时显著降低U251细胞的增殖能力,促进肿瘤细胞凋亡,表明CDKL1基因可能具有影响胶质瘤发生、发展的作用。
Objective To silence the expression of cyclin-dependent kinase-like 1 (CDKL1) gene in human glioma U251 cells by RNA interference (RNAi) and investigate its effect on the proliferation and apoptosis of tumor cells. Methods The shRNA lentiviral expression vector targeting CDKL1 gene was constructed and packaged into viral particles and infected with glioma U251 cells. The mRNA and protein expression of CDKL1 gene was verified by real-time quantitative polymerase chain reaction and Western blotting. Determine its inhibitory efficiency. Cell proliferation and apoptosis were detected by methylthiazolyl tetrazolium (MTT) assay and Annexin V staining. The effect of silencing CDKL1 gene on the proliferation and apoptosis phenotype of U251 cells was observed. Results CDKL1-shRNA effectively inhibited the expression of CDKL1 mRNA and protein in U251 cells. After CDKL1 gene silencing, the optical density values of U251 in each phase decreased compared with those in non-sense sequences. The difference was statistically significant on the 5th day (P <0.05). At the same time, the percentage of apoptosis in U251 cells that up-regulated the expression of CDKL1 increased from (9.80 ± 0.21)% to (19.26 ± 0.33)% in non-small interfering RNA (siRNA) control group, with statistical significance (P <0.05). Conclusion The RNAi method can effectively silence the expression of CDKL1 gene, significantly reduce the proliferation of U251 cells and promote the apoptosis of tumor cells, indicating that CDKL1 gene may have an effect on the occurrence and development of glioma.