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Ascl2 knockdown results in tumor growth arrest by miRNA-302b-related inhibition of colon cancer progenitor cells.

Zhu R, Yang Y, Tian Y, Bai J, Zhang X, Li X, Peng Z, He Y, Chen L, Pan Q, Fang D, Chen W, Qian C, Bian X, Wang R - PLoS ONE (2012)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT

Background: 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.

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Related in: MedlinePlus

The percentage of CD133+ HT-29 cells and the expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vitro.54.7% of shRNA-Ctr/HT-29 cells are positive for CD133 expression compared with 26.2% of HT-29 cells are positive for CD133 expression in shRNA-Ascl2/HT-29 cells (*: p<0.05) (A and B). The mRNA levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Lgr5, Oct4, Bmi1, Sox2, and C-myc analyzed by real-time PCR in the shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (*: p<0.05; **: p<0.01) (C). The protein levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than that in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (D).
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pone-0032170-g005: The percentage of CD133+ HT-29 cells and the expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vitro.54.7% of shRNA-Ctr/HT-29 cells are positive for CD133 expression compared with 26.2% of HT-29 cells are positive for CD133 expression in shRNA-Ascl2/HT-29 cells (*: p<0.05) (A and B). The mRNA levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Lgr5, Oct4, Bmi1, Sox2, and C-myc analyzed by real-time PCR in the shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (*: p<0.05; **: p<0.01) (C). The protein levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than that in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (D).

Mentions: The observation that Ascl2 expression was higher in CD133+ HT-29 cells than in CD133− HT-29 cells, led to the hypothesis that the Ascl2 protein is important for the expression of CD133 in CD133+ stem/progenitor HT-29 cells. The comparative flow cytometry analysis of shRNA-Ctr/HT-29 and shRNA-Ascl2/HT-29 cells was performed. In the shRNA-Ctr/HT-29 cells, 54.7% of the cells were positive for CD133 expression, whereas 26.2% of the cells were positive for CD133 expression in shRNA-Ascl2/HT-29 cells (Figure 5A). Triple experiments, using three different clones, confirmed that the percentage of CD133+ HT-29 cells was significantly reduced in the presence of Ascl2 knockdown (Figure 5B) (p<0.05). Only 0.1% of LS174T cells were CD133 positive in our own data and 0.45% of LS174T cells were CD133 positive in literarure [20] with flow cytometry sorting, so the comparative flow cytometry analysis of shRNA-Ctr/LS174T and shRNA-Ascl2/LS174T cells was not performed.


Ascl2 knockdown results in tumor growth arrest by miRNA-302b-related inhibition of colon cancer progenitor cells.

Zhu R, Yang Y, Tian Y, Bai J, Zhang X, Li X, Peng Z, He Y, Chen L, Pan Q, Fang D, Chen W, Qian C, Bian X, Wang R - PLoS ONE (2012)

The percentage of CD133+ HT-29 cells and the expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vitro.54.7% of shRNA-Ctr/HT-29 cells are positive for CD133 expression compared with 26.2% of HT-29 cells are positive for CD133 expression in shRNA-Ascl2/HT-29 cells (*: p<0.05) (A and B). The mRNA levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Lgr5, Oct4, Bmi1, Sox2, and C-myc analyzed by real-time PCR in the shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (*: p<0.05; **: p<0.01) (C). The protein levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than that in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285660&req=5

pone-0032170-g005: The percentage of CD133+ HT-29 cells and the expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vitro.54.7% of shRNA-Ctr/HT-29 cells are positive for CD133 expression compared with 26.2% of HT-29 cells are positive for CD133 expression in shRNA-Ascl2/HT-29 cells (*: p<0.05) (A and B). The mRNA levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Lgr5, Oct4, Bmi1, Sox2, and C-myc analyzed by real-time PCR in the shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (*: p<0.05; **: p<0.01) (C). The protein levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than that in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (D).
Mentions: The observation that Ascl2 expression was higher in CD133+ HT-29 cells than in CD133− HT-29 cells, led to the hypothesis that the Ascl2 protein is important for the expression of CD133 in CD133+ stem/progenitor HT-29 cells. The comparative flow cytometry analysis of shRNA-Ctr/HT-29 and shRNA-Ascl2/HT-29 cells was performed. In the shRNA-Ctr/HT-29 cells, 54.7% of the cells were positive for CD133 expression, whereas 26.2% of the cells were positive for CD133 expression in shRNA-Ascl2/HT-29 cells (Figure 5A). Triple experiments, using three different clones, confirmed that the percentage of CD133+ HT-29 cells was significantly reduced in the presence of Ascl2 knockdown (Figure 5B) (p<0.05). Only 0.1% of LS174T cells were CD133 positive in our own data and 0.45% of LS174T cells were CD133 positive in literarure [20] with flow cytometry sorting, so the comparative flow cytometry analysis of shRNA-Ctr/LS174T and shRNA-Ascl2/LS174T cells was not performed.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT

Background: 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.

Show MeSH
Related in: MedlinePlus