RNA干扰抑制CD44和CD133的表达对肺腺癌A549细胞增殖能力的影响

目的 观察CD44和CD133的表达对人肺腺癌A549细胞株增殖能力的影响.方法 筛选可持续增殖的A549细胞并培养,按CD44和133表达的不同将筛选出的细胞分成5组,非转染组:CD44+CD133+细胞(A组)、CD44-/CD133-细胞(B组)、未分选A549细胞(C组);转染组:CD44-和CD133-细胞(D组)及阴性对照组(E组).筛选得到沉默效果最佳的小干扰RNA(siRNA)片段,并用其转染各组细胞,采用MTS法检测并比较干扰前后各组细胞的增殖能力.结果　沉默效果最佳的siRNA片段及浓度为siCD44-h_002 25 nmol/L和siCD133-h_002 25 nmol/L,沉默效率分别为62%和82%.用其转染各组样本后,CD44和CD133的表达明显被抑制,比较转染前后各组样本的灰度值,差异有统计学意义(P＜0.01).通过分析MTS图显示,转染后细胞的增殖能力显著下降.结论　CD44和CD133的阳性表达,与人肺腺癌A549细胞株较强的增殖能力有关,CD44和CD133可能是肺腺癌干细胞的表面标志物.

Abstract：

Objective To study the effect of expression of CD44 and CD133 on the tumorigenesis of human lung adenocarcinoma cell line A549. Methods A549 cells having continuous self-renewal ability were cultured, and classified into 5 subpopulations based on differential expression patterns for CD133and CD44. The small interfering RNA (siRNA) with optimal sequences was screened out, and transfected the A549 cells. The methods of MTS and cell counting were used to investigate the proliferation of A549cells before and after the expression of CD133 and CD44 was inhibited by RNAi. Results For the optimal sequences and concentrations for siRNA transfection efficiency being 25 nmol/L for both siCD44-h_002 25and siCD133-h_002 25, the transfection efficiency was 62% and 82% respectively. Stably transfected cells were screened, and the expression of CD44 and CD133 was inhibited significantly. The gray values before transfection were significantly different from those after transfection ( P ＜ 0. 01 ). The MTS curve showed that the cell viability was significantly decreased after transfection. Conclusion The proliferation ability of A549 cells was related to the positive expression of CD44 and CD133. CD133 and CD44 may be important surface markers of lung cancer stem cells.

Effect of RNAi inhibition to expression of CD44 and CD133 on tumorigenesis of human lung adenocarcinoma cell line A549

Objective To study the effect of expression of CD44 and CD133 on the tumorigenesis of human lung adenocarcinoma cell line A549. Methods A549 cells having continuous self-renewal ability were cultured, and classified into 5 subpopulations based on differential expression patterns for CD133and CD44. The small interfering RNA (siRNA) with optimal sequences was screened out, and transfected the A549 cells. The methods of MTS and cell counting were used to investigate the proliferation of A549cells before and after the expression of CD133 and CD44 was inhibited by RNAi. Results For the optimal sequences and concentrations for siRNA transfection efficiency being 25 nmol/L for both siCD44-h_002 25and siCD133-h_002 25, the transfection efficiency was 62% and 82% respectively. Stably transfected cells were screened, and the expression of CD44 and CD133 was inhibited significantly. The gray values before transfection were significantly different from those after transfection ( P ＜ 0. 01 ). The MTS curve showed that the cell viability was significantly decreased after transfection. Conclusion The proliferation ability of A549 cells was related to the positive expression of CD44 and CD133. CD133 and CD44 may be important surface markers of lung cancer stem cells.