Human colon cancer HT-29 and LS174T cells: stably transfected with shRNA-Ascl2/EGFP vs. stably transfected with shRNA-Control/EGFP. Homo sapiens

Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell lines HT-29 (47.5-95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. The microRNA (miRNA) microarrays identified 26 two-fold up-regulated miRNAs and 58 two-fold down-regulated miRNAs in shRNA-Ascl2/HT-29 cells and 178 two-fold up-regulated miRNAs and 172 two-fold down-regulated miRNAs in shRNA-Ascl2/LS174T cells. Overall design: Two-condition experiment: shRNA-Ascl2/HT-29 cells vs. shRNA-Ctr/HT-29 cells, and shRNA-Ascl2/LS174T cells vs. shRNA-Ctr/LS174T cells. Biological replicates: 1 HT-29 cells stably transfected with shRNA-Ascl2/EGFP, 1 LS174T cells stably transfected with shRNA-Ascl2/EGFP, 1 HT-29 cells stably transfected with shRNA-Control/EGFP, and 1 LS174T cells stably transfected with shRNA-Control/EGFP, independently grown and harvested. One replicate per array.

Achaete scute样蛋白2（Ascl2）是一种碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）转录因子，可调控肠道干细胞的命运。然而，Ascl2在结肠癌祖细胞中的功能仍未明确。研究选取HT-29细胞（CD133阳性群体占比47.5%~95%）与LS174T细胞（CD133阳性群体占比0.45%），通过RNA干扰（RNA interference）敲低Ascl2基因后，对其在结肠癌祖细胞中的功能进行评价。通过microRNA（miRNA）微阵列检测发现，在shRNA-Ascl2/HT-29细胞中，存在26种上调2倍的miRNA与58种下调2倍的miRNA；在shRNA-Ascl2/LS174T细胞中，则分别存在178种上调2倍的miRNA与172种下调2倍的miRNA。实验总体设计：本实验为双条件对照试验，分别为shRNA-Ascl2/HT-29细胞与shRNA对照转染的HT-29（shRNA-Ctr/HT-29）细胞的比较，以及shRNA-Ascl2/LS174T细胞与shRNA对照转染的LS174T（shRNA-Ctr/LS174T）细胞的比较。生物学重复设置如下：分别构建1株稳定转染shRNA-Ascl2/EGFP的HT-29细胞、1株稳定转染shRNA-Ascl2/EGFP的LS174T细胞、1株稳定转染shRNA对照/EGFP（shRNA-Control/EGFP）的HT-29细胞与1株稳定转染shRNA对照/EGFP的LS174T细胞，各株细胞独立培养并收获；每块阵列对应一个生物学重复。