RNA干扰沉默CDKL1基因对人胶质瘤U251细胞增殖和凋亡的影响

Effect of silencing cyclin-dependent kinase-like 1 gene by RNA interference on proliferation and apoptosis in human U251 glioma cells

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 investigate the effect of silencing cyclin-dependent kinase-like I（CDKL1） gene by RNA interfering on proliferation and apoptosis in human U251 glioma cells. Methods Small interference RNA （siRNA） targeting CDKL1 gene was cloned into lentivirus vector. Then shRNA lentivirus particles were packaged and infected into U251 cells. Real-time polymerase chain reaction （PCR） and Western blotting analysis were performed to investigate the expression of CDKL1 at mRNA and protein levels. The cell proliferation and apoptosis were detected by methyl thiazolyl tetrazolium （MTT） assay and flow cytometry marked with Annexin V. Results Real-time PCR and Western blotting analysis showed that U251 cells infected by shRNA lentivirus had significantly down-regulated expression of CDKL1 mRNA and protein. MTT assay showed that cell proliferation of U251 cells with CDKL1 silenced was suppressed from day 1 to 4, and significantly decreased on day 5 compared with the nonsilenced group（ P 〈0. 05）. The apoptosis rate increased from （9. 80 ±0. 21 ）% in the non-silenced group to （19.26± 0.33）% in the silenced group as detected by flow cytometry （P〈0.05）. Conclusion The lentivirus vector harboring shRNA targeting CDKL1 gene can successfully down-regulate CDKL1 mRNA and protein in U251 cells. And CDKL1 silencing can inhibit proliferation and enhance apoptosis in U251 cells, indicating that CDKL1 can promote the development and progression of human glioma. （Shanghai Med J, 2011, 34：382-385）