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TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways

View Article: PubMed Central - PubMed

ABSTRACT

Background: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/β-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce.

Methods: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR.

Results: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1.

Conclusion: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13046-016-0426-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Knock-down of TLE3 promoted the proliferation and tumorigenesis of human CRC cells. a RNAi-silencing of TLE3 in shRNA-transduced stable HCT15 and SW620 cells. α-Tubulin was used as a loading control. b Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by MTT assays. c Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by colony formation assays. d Knock-down of TLE3 promoted anchorage independent growth ability of HCT15 and SW620 cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW620/Scr and SW620/sh-1 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
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Fig3: Knock-down of TLE3 promoted the proliferation and tumorigenesis of human CRC cells. a RNAi-silencing of TLE3 in shRNA-transduced stable HCT15 and SW620 cells. α-Tubulin was used as a loading control. b Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by MTT assays. c Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by colony formation assays. d Knock-down of TLE3 promoted anchorage independent growth ability of HCT15 and SW620 cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW620/Scr and SW620/sh-1 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05

Mentions: To further confirm the role of TLE3 in human CRC cells proliferation, endogenous expression of TLE3 in HCT15 and SW620 was knocked down by specific shRNAs (Fig. 3a). MTT and colony formation assays indicated that knock-down of TLE3 expression obviously promoted the cell growth of HCT15 and SW620 cells in comparison with control cells (Fig. 3b and c). Besides, the number and size of colonies in soft agar assays were significantly increased in TLE3-silenced HCT15 and SW620 cells in comparison with control cells (Fig. 3d). Furthermore, knockdown of endogenous TLE3 expression in SW620 cells led to noteworthy promotion of tumor growth and volume in the tumorigenesis assays by subcutaneous injection in nude mice, confirming the suppressive effect of TLE3 on CRC proliferation in vivo (Fig. 3e; n = 6). In contrast to tumors of TLE3 overexpression, Ki-67 index was found much higher in tumors of TLE3 knock-down in comparison with control cell-based tumor (Fig. 3f).Fig. 3


TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways
Knock-down of TLE3 promoted the proliferation and tumorigenesis of human CRC cells. a RNAi-silencing of TLE3 in shRNA-transduced stable HCT15 and SW620 cells. α-Tubulin was used as a loading control. b Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by MTT assays. c Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by colony formation assays. d Knock-down of TLE3 promoted anchorage independent growth ability of HCT15 and SW620 cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW620/Scr and SW620/sh-1 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037636&req=5

Fig3: Knock-down of TLE3 promoted the proliferation and tumorigenesis of human CRC cells. a RNAi-silencing of TLE3 in shRNA-transduced stable HCT15 and SW620 cells. α-Tubulin was used as a loading control. b Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by MTT assays. c Knock-down of TLE3 promoted cell proliferation of HCT15 and SW620 cells by colony formation assays. d Knock-down of TLE3 promoted anchorage independent growth ability of HCT15 and SW620 cells as determined by soft agar assays. Colonies containing more than 50 cells were scored. e Tumorigenesis assay by subcutaneous injection of SW620/Scr and SW620/sh-1 cells in nude mice (n = 6/group). Tumor volumes were measured on the indicated days. Data points are the mean tumor volumes ± SD. f The sections of tumor were under H&E staining or subjected to IHC staining using an antibody against Ki-67. Error bars represent the mean ± SD of 3 independent experiments. * p < 0.05
Mentions: To further confirm the role of TLE3 in human CRC cells proliferation, endogenous expression of TLE3 in HCT15 and SW620 was knocked down by specific shRNAs (Fig. 3a). MTT and colony formation assays indicated that knock-down of TLE3 expression obviously promoted the cell growth of HCT15 and SW620 cells in comparison with control cells (Fig. 3b and c). Besides, the number and size of colonies in soft agar assays were significantly increased in TLE3-silenced HCT15 and SW620 cells in comparison with control cells (Fig. 3d). Furthermore, knockdown of endogenous TLE3 expression in SW620 cells led to noteworthy promotion of tumor growth and volume in the tumorigenesis assays by subcutaneous injection in nude mice, confirming the suppressive effect of TLE3 on CRC proliferation in vivo (Fig. 3e; n = 6). In contrast to tumors of TLE3 overexpression, Ki-67 index was found much higher in tumors of TLE3 knock-down in comparison with control cell-based tumor (Fig. 3f).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/&beta;-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce.

Methods: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR.

Results: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1.

Conclusion: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.

Electronic supplementary material: The online version of this article (doi:10.1186/s13046-016-0426-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus