| Background: Colon cancer is one of the common gastrointestinal cancer, with poorprognosis and a higher mortality rate. Treatment of colon cancer has been a major problem,because its early diagnosis rate is low, and treatment of advanced colon cancer withinvasion and metastasis faces enormous challenges. The cancer stem cell theory hasbrought new hope for the treatment of colon cancer. The theory is that cancer stem cells area class of seed cells, with self-renewal and multilineage differentiation potential, and theyare a minority of all tumor cells. But because of their unlimited proliferation andasymmetric division, cancer stem cells are roots of malignant tumor growth, invasion,metastasis and recurrence. Cancer stem cells are the seeds and sources of tumors, they are"initiating cells" or "power cells" in the process of tumor development, metastasis andrecurrence, they play a key role in all tumor processes. The current studies suggest thatcolon cancer stem cells may have originated in the normal adult intestinal crypt stem cells.Achaete scute-like2(Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controlsthe fate of intestinal stem cells. Ascl2transgenic overexpression in mouse inducedintestinal epithelial crypt hyperplasia and ectopic crypts in the intestinal villi; Ascl2geneknockdown in adult mouse intestinal led to disappear of Lgr5positive adult intestinal cryptstem cells within a few days. And, recent studies have found that the expression level ofAscl2is upregulated in human colon cancer specimens. Therefore, we assume that, Ascl2may be associated with colon cancer stem/progenitor cells. However, the role of Ascl2incolon 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 functionalevaluation of Ascl2in colon cancer progenitor cells after gene knockdown by RNAinterference.Methodology/Principle Findings: Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas, and Ascl2expression wasspecifically localized at the nucleus of crypt base cells of normal human colon mucosa.Downregulation of Ascl2using RNA interference in cultured colonic adenocarcinomaHT-29and LS174T cells reduced cellular proliferation, colony-forming ability, invasion andmigration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2protein level in CD133+HT-29cells was significantly higher than in CD133-HT-29cells.Ascl2blockade via shRNA interference in HT-29cells (shRNA-Ascl2/HT-29cells)resulted in26.2%of cells staining CD133+compared with54.7%in controlshRNA-Ctr/HT-29cells. The levels of 'stemness' associated genes, such as CD133, Sox2,Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29andshRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft(CD133was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29cellshad inhibited abilities to form tumorspheres compared with control. The microRNA(miRNAs) microarrays, identified26up-regulated miRNAs and58down-regulatedmiRNAs in shRNA-Ascl2/HT-29cells. 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 ofmiRNA-302b, via its mimics, 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 andmigration in vitro.Conclusions/Significance: Ascl2RNA interference can lead to growth inhibition ofcolon cancer cells. Ascl2is an important transcription factor closely related to colon cancerstem/progenitor cells, and functions through a miR-302b-related mechanism. Ascl2maybe a potential target for treatment of colon cancer.
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