miR-21对人肺癌细胞NCI-H446凋亡的影响

Effects of miR-21 on apoptosis of human lung cancer NCI-H446 cells

目的探讨微小RNA-21(microRNA-21,miR-21)对人肺癌细胞NCI-H446凋亡的影响。方法培养肺癌细胞株NCI-H446,分别以反义寡聚核苷酸miR-21(antisense oligonucleotide miR-21,as-miR-21)转染沉默miR-21(miR-21 ASO转染组)、转染无关乱码寡核苷酸序列(阴性对照组),以及不转染寡核苷酸(空白对照组)。采用实时荧光定量聚合酶链反应法(real-time quantitative PCR,qRT-PCR)检测各组miR-21的表达变化;以流式细胞术和Hochest 33258荧光染色检测三组细胞的凋亡情况,并用免疫印迹法(Western blot)检测凋亡相关蛋白B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)表达水平变化。结果用以as-miR-21转染沉默miR-21后NCI-H446细胞中miR-21表达水平低于阴性对照组和空白对照组(均P<0.05);流式细胞术结果显示,用as-miR-21转染NCI-H446细胞后其细胞凋亡率明显高于阴性对照组和空白组(均P<0.05),凋亡率分别为(12.82±0.01)%、(5.23±0.01)%和(4.34±0.01)%;Hochest 33258荧光染色显示,miR-21 ASO转染组、阴性对照组和空白对照组细胞凋亡指数分别为(8.37±1.35)%、(3.12±1.12)%和(2.56±3.43)%,转染组与其他两组比较差异有统计学意义(均P<0.05);Western blot检测发现miR-21 ASO转染组的Bcl-2表达与阴性对照组和空白对照组比较,明显降低(均P<0.05)。结论下调肺癌细胞NCI-H446中miR-21的表达能促进肺癌细胞的凋亡,其作用机制可能是通过下调Bcl-2的表达而实现的。

Objective To investigate the effects of microRNA-21（ miR-21） on the apoptosis of human lung cancer NCI-H446 cells. Methods Cultured lung cancer NCI-H446 cells were transfected with antisense oligonucleotide miR-21（ as-miR-21） or non-special oligonucleotides（ negative control） and non-transfected cells were used as blank control. miR-21 expression were determined by fluorescence real-time quantitative PCR method; The apoptosis rate of NCI-H446 cells was measured by flow cytometry and Hochest 33258; B-cell lymphoma-2（ Bcl-2） protein was detected by Western blot. Results The expression of miR-21 in as-miR-21 group was significantly lower than those in negative and blank control groups（ both P〈 0. 05）. The apoptosis rate in as-miR-21 transfected cells was significantly higher than those in negative control and blank control groups [flow cytometry：（ 12. 82 ± 0. 01） % vs（ 5. 23 ± 0. 01） %,（ 4. 34 ± 0. 01） %,both P〈 0. 05;Hochest 33258：（ 8. 37 ± 1. 35） % vs（ 3. 12 ± 1. 12） %,（ 2. 56 ± 3. 43） %,both P〈 0. 05]. The expression of Bcl-2 protein in as-miR-21 group was significantly lower than that in negative and blank control groups（ both P〈 0. 05）. Conclusion Down-regulation of miR-21 expression in NCI-H446 cells promotes cell apoptosis,which may be associated with the downregulation of Bcl-2 expression.