Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.
PLoS One. 2012;7(2):e32170. doi: 10.1371/journal.pone.0032170. Epub 2012 Feb 23.
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 line 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.
METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133(+) HT-29 cells was significantly higher than in CD133(-) HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133(+) compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of 'stemness' associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of 'stemness', were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 'stemness' characteristics, including tumorsphere formation and 'stemness' associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro.
CONCLUSIONS/SIGNIFICANCE: Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.
Achaete scute-like 2（Ascl2），一种基本螺旋-环-螺旋（bHLH）转录因子，控制肠道干细胞的命运。然而，Ascl2 在结肠癌细胞祖细胞中的作用仍不清楚。选择 HT-29（CD133（+）群体的 47.5-95％）和 LS174T（CD133（+）群体的 0.45％）细胞系，通过 RNA 干扰基因敲低来评估 Ascl2 在结肠癌细胞祖细胞中的功能。
方法/主要发现：免疫组织化学显示结直肠腺癌中 Ascl2 显著增加。用 RNA 干扰下调培养的结肠腺癌 HT-29 和 LS174T 细胞中的 Ascl2，减少了细胞增殖、体外集落形成能力、侵袭和迁移，并导致体内肿瘤异种移植物的生长停滞。CD133（+）HT-29 细胞中的 Ascl2 蛋白水平明显高于 CD133（-）HT-29 细胞。在 HT-29 细胞中通过 shRNA 干扰阻断 Ascl2（shRNA-Ascl2/HT-29 细胞）导致 26.2％的细胞染色 CD133（+），而对照 shRNA-Ctr/HT-29 细胞为 54.7％。体外以及相应的肿瘤异种移植中，与对照相比，shRNA-Ascl2/HT-29 和 shRNA-Ascl2/LS174T 细胞中与“干性”相关的基因（如 CD133、Sox2、Oct4、Lgr5、Bmi1 和 C-myc）水平显著降低。与对照相比，shRNA-Ascl2/HT-29 细胞形成肿瘤球的能力受到抑制。miRNA 微阵列鉴定出 shRNA-Ascl2/HT-29 细胞中 26 个上调的 miRNA 和 58 个下调的 miRNA。通过 qPCR 定量检测了涉及“干性”调节的 let-7b、miRNA-124、miRNA-125b、miRNA-17、miRNA-20a 和 miRNA-302b 的表达水平，证实了它们的身份。通过其模拟物恢复 miRNA-302b，导致 shRNA-Ascl2/HT-29“干性”特征的恢复，包括肿瘤球形成和与“干性”相关的基因水平，以及细胞行为的恢复，包括体外集落形成能力、侵袭和迁移。
结论/意义：Ascl2 可能是抑制结肠癌细胞祖细胞的潜在靶点，其功能通过 miR-302b 相关机制实现。
