Abstract
BackgroundAchaete 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 FindingsImmunohistochemistry 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/SignificanceAscl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.
Highlights
Colorectal cancer (CRC), the third leading cause of death from cancer worldwide and a leading cause of morbidity and mortality in developed countries [1], represents a major therapeutic challenge for cancer
We demonstrate the selective blockade of Achaete scute-like 2 (Ascl2) in HT-29 and LS174T cells could inhibit cell growth, invasion and migration in vitro, and lead to growth arrest in vivo, that is partially related to miRNA-302b-related inhibition of ‘stemness’ of colon cancer progenitor cells based on the experiments of shRNAAscl2/HT-29 transfected with miRNA-302b mimic
Ascl2 is overexpressed in colon cancer and colon cancer cell lines, and Ascl2 interference in HT-29 and LS174T cells remarkably reduced its expression
Summary
Colorectal cancer (CRC), the third leading cause of death from cancer worldwide and a leading cause of morbidity and mortality in developed countries [1], represents a major therapeutic challenge for cancer. The cancer stem cell (CSC) hypothesis has been proposed to explain the functional heterogeneity and carcinogenesis of cancer. According to this model, a subpopulation of cancer cells, which exhibit stem-like features, sustain tumor formation, metastasis, and resistance to therapy [2,3,4,5]. A subpopulation of cancer cells, which exhibit stem-like features, sustain tumor formation, metastasis, and resistance to therapy [2,3,4,5] In this respect, CSCs would be expected to have a stem celllike/progenitor phenotype (generally referred to as ‘‘stemness’’). The cell line HT-29 (47.5–95% of CD133+ population) and LS174T (0.45% of CD133+ population) were chosen for functional evaluation of Ascl in colon cancer progenitor cells after gene knockdown by RNA interference
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