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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

Evaluation of TLE3 expression in CRC cell lines and primary human CRC. a Analyses of TLE3 expression in normal colorectal intestines (n = 41) and colorectal tumors (n = 465) in The Cancer Genome Atlas (TCGA) microarray dataset. b Analyses of TLE3 expression in 41 pairs of tissues of normal colorectal intestines and colorectal tumors in TCGA microarray dataset. c Expression of TLE3 protein in 8 primary human CRC tissue (T) and the adjacent normal tissue (N) paired from the same patient by Western blot. d Real-time PCR was used to quantify average N/T ratios of TLE3 mRNA expression. e Representative images of TLE3 protein expression in normal colorectal epithelia, adenoma and adenocarcinoma by IHC. TLE3 was positively detected in both adenoma and their adjacent normal tissue (middle and left), whereas there was little detection in adenocarcinoma (right). f Influence of TLE3 expression on overall survival by Kaplan-Meier analysis in 105 patients with CRC. p < 0.05 The expression levels of protein or mRNA were normalized with α-Tubulin or GAPDH, respectively. Error bars represent mean ± SD calculated from 3 independent experiments
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Fig1: Evaluation of TLE3 expression in CRC cell lines and primary human CRC. a Analyses of TLE3 expression in normal colorectal intestines (n = 41) and colorectal tumors (n = 465) in The Cancer Genome Atlas (TCGA) microarray dataset. b Analyses of TLE3 expression in 41 pairs of tissues of normal colorectal intestines and colorectal tumors in TCGA microarray dataset. c Expression of TLE3 protein in 8 primary human CRC tissue (T) and the adjacent normal tissue (N) paired from the same patient by Western blot. d Real-time PCR was used to quantify average N/T ratios of TLE3 mRNA expression. e Representative images of TLE3 protein expression in normal colorectal epithelia, adenoma and adenocarcinoma by IHC. TLE3 was positively detected in both adenoma and their adjacent normal tissue (middle and left), whereas there was little detection in adenocarcinoma (right). f Influence of TLE3 expression on overall survival by Kaplan-Meier analysis in 105 patients with CRC. p < 0.05 The expression levels of protein or mRNA were normalized with α-Tubulin or GAPDH, respectively. Error bars represent mean ± SD calculated from 3 independent experiments

Mentions: In order to identify deregulated genes involved in the progression of CRC, gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray datasets. The analyses showed that TLE3 was significantly down-regulated in 465 CRC tissues compared to 41 normal tissues (Fig. 1a and b). Consistent with this finding, Western blot and real-time PCR analyses showed that TLE3 expression was significantly down-regulated in eight CRC tissues compared with adjacent normal intestine epithelial tissues (Fig. 1c and d). Furthermore, TLE3 protein expression was detected by immunohistochemistry (IHC) in 105 paraffin-embedded, archived human CRC tissues. TLE3 protein expression was quite abundant in adenoma as well as normal tissue, whereas it was markedly decreased in adenocarcinoma (Fig. 1e). Kaplan-Meier survival analysis showed that CRC patients with lower level of TLE3 protein expression had a poorer prognosis (Fig. 1f). These results suggest that TLE3 down-regulation is significantly associated with advanced progression of human CRC.Fig. 1


TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways
Evaluation of TLE3 expression in CRC cell lines and primary human CRC. a Analyses of TLE3 expression in normal colorectal intestines (n = 41) and colorectal tumors (n = 465) in The Cancer Genome Atlas (TCGA) microarray dataset. b Analyses of TLE3 expression in 41 pairs of tissues of normal colorectal intestines and colorectal tumors in TCGA microarray dataset. c Expression of TLE3 protein in 8 primary human CRC tissue (T) and the adjacent normal tissue (N) paired from the same patient by Western blot. d Real-time PCR was used to quantify average N/T ratios of TLE3 mRNA expression. e Representative images of TLE3 protein expression in normal colorectal epithelia, adenoma and adenocarcinoma by IHC. TLE3 was positively detected in both adenoma and their adjacent normal tissue (middle and left), whereas there was little detection in adenocarcinoma (right). f Influence of TLE3 expression on overall survival by Kaplan-Meier analysis in 105 patients with CRC. p < 0.05 The expression levels of protein or mRNA were normalized with α-Tubulin or GAPDH, respectively. Error bars represent mean ± SD calculated from 3 independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037636&req=5

Fig1: Evaluation of TLE3 expression in CRC cell lines and primary human CRC. a Analyses of TLE3 expression in normal colorectal intestines (n = 41) and colorectal tumors (n = 465) in The Cancer Genome Atlas (TCGA) microarray dataset. b Analyses of TLE3 expression in 41 pairs of tissues of normal colorectal intestines and colorectal tumors in TCGA microarray dataset. c Expression of TLE3 protein in 8 primary human CRC tissue (T) and the adjacent normal tissue (N) paired from the same patient by Western blot. d Real-time PCR was used to quantify average N/T ratios of TLE3 mRNA expression. e Representative images of TLE3 protein expression in normal colorectal epithelia, adenoma and adenocarcinoma by IHC. TLE3 was positively detected in both adenoma and their adjacent normal tissue (middle and left), whereas there was little detection in adenocarcinoma (right). f Influence of TLE3 expression on overall survival by Kaplan-Meier analysis in 105 patients with CRC. p < 0.05 The expression levels of protein or mRNA were normalized with α-Tubulin or GAPDH, respectively. Error bars represent mean ± SD calculated from 3 independent experiments
Mentions: In order to identify deregulated genes involved in the progression of CRC, gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray datasets. The analyses showed that TLE3 was significantly down-regulated in 465 CRC tissues compared to 41 normal tissues (Fig. 1a and b). Consistent with this finding, Western blot and real-time PCR analyses showed that TLE3 expression was significantly down-regulated in eight CRC tissues compared with adjacent normal intestine epithelial tissues (Fig. 1c and d). Furthermore, TLE3 protein expression was detected by immunohistochemistry (IHC) in 105 paraffin-embedded, archived human CRC tissues. TLE3 protein expression was quite abundant in adenoma as well as normal tissue, whereas it was markedly decreased in adenocarcinoma (Fig. 1e). Kaplan-Meier survival analysis showed that CRC patients with lower level of TLE3 protein expression had a poorer prognosis (Fig. 1f). These results suggest that TLE3 down-regulation is significantly associated with advanced progression of human CRC.Fig. 1

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