Li Zhongjun, Xiang Yang, Xiang Lixin, Xiao Yanni, Li Fengjie, Hao Ping
Department of Blood Transfusion, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.
Department of Oncology, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.
PLoS One. 2014 Mar 26;9(3):e92669. doi: 10.1371/journal.pone.0092669. eCollection 2014.
To evaluate the expression of ALDH1A1 in lung adenoma stem cells (LASCs) and maintenance of their stemness through the Notch pathway.
LASCs (A549s) were isolated from lung adenoma cells (A549) and identified by their coexpression of CD133 and CD326 and their capacity formulti-directional differentiation. Expression of ALDH1A1 in A549 and A549s cells were evaluated by Real-time PCR. Effects of ALDH1A1 upregulation in A549 cells and its downregulation in A549s cells on the clonogenicity and cell cycle were assessed by colony-forming unit assay. Moreover, the effects of ALDH1A1 on the Notch pathway, and thus on the cell cycle, were studied.
A549s cells were successfully isolated and identified.ALDH1A1expression was significantly higher in A549s than in A549 cells. Clonogenicity was significantly decreased in A549s cells treated with ALDH1A1 siRNA. Duration of the G1 stage of the cell cycle increased after ALDH1A1 was overexpressed, or decreased with ALDH1A1 siRNA. ALDH1A1, Notch1, -2, and -3, CDK2, and CCNE1 expression levels were higher in A549s cells than in A549 cells. Expression of Notch1, -2, and -3, CDK2, and CCNE1 was significantly decreased by upregulation of ALDH1A1 in A549 cells, but increased by its interruption in A549s cells. When Notch3 or CDK2 expression was downregulated, the expression levels of ALDH1A1, Notch1, -2, and -3, CDK2, and CCNE1 were reduced in all cell types.
ALDH1A1 expression improved clonogenicity and inhibited the cell cycle, maintaining the stemness of the A549s cells; this may involve suppression of the Notch/CDK2/Cyclin pathway.
评估醛脱氢酶1A1（ALDH1A1）在肺腺癌干细胞（LASC）中的表达情况，以及其通过Notch信号通路维持干细胞特性的作用。
从肺腺癌细胞（A549）中分离出LASC（A549s），并通过共表达CD133和CD326以及多向分化能力进行鉴定。采用实时定量PCR评估A549和A549s细胞中ALDH1A1的表达。通过集落形成单位试验评估A549细胞中ALDH1A1上调和A549s细胞中ALDH1A1下调对克隆形成能力和细胞周期的影响。此外，研究了ALDH1A1对Notch信号通路进而对细胞周期的影响。
成功分离并鉴定出A549s细胞。A549s细胞中ALDH1A1的表达明显高于A549细胞。用ALDH1A1小干扰RNA处理的A549s细胞克隆形成能力显著降低。过表达ALDH1A1后细胞周期G1期持续时间增加，而用ALDH1A1小干扰RNA处理则使其缩短。A549s细胞中ALDH1A1、Notch1、-2和-3、细胞周期蛋白依赖性激酶2（CDK2）以及细胞周期蛋白E1（CCNE1）的表达水平高于A549细胞。在A549细胞中上调ALDH1A1可使Notch1、-2和-3、CDK2以及CCNE1的表达显著降低，而在A549s细胞中干扰ALDH1A1则使其表达增加。当Notch3或CDK2表达下调时，所有细胞类型中ALDH1A1、Notch1、-2和-3、CDK2以及CCNE1的表达水平均降低。
ALDH1A1的表达提高了克隆形成能力并抑制细胞周期，维持了A549s细胞的干细胞特性；这可能涉及对Notch/CDK2/细胞周期蛋白通路的抑制。
