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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

TLE3 inhibited cell cycle progression of human CRC cell. a, b Representative figures depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. c, d Flow cytometry of annexin V and PI-labelled CRC cells indicating apoptosis
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Fig4: TLE3 inhibited cell cycle progression of human CRC cell. a, b Representative figures depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. c, d Flow cytometry of annexin V and PI-labelled CRC cells indicating apoptosis

Mentions: To explore the possible mechanism by which TLE3 regulates the proliferation of human CRC cells, we analyzed TLE3 RNA expression levels based on TCGA COAD RNA Seq dataset and cycling gene signatures from the online Gene Set Database of the gene set enrichment analysis (GSEA) [34]. We observed that TLE3 expression was negatively correlated with genes related to cell cycle and G1-S transition (Additional file 2: Figure S1a and b). Furthermore, flow cytometry was performed to measure the distribution of cell cycle phases. Compared with control cells, the percentage of G1-phase cells increased and S-phase decreased significantly in the SW480 cells with TLE3 overexpressing (27.14 vs 41.55 %, p < 0.05, and 49.47 vs 34.02 %, p < 0.05, respectively) (Fig. 4a and Additional file 3: Figure S2a). Another cell line Ls174t of TLE3 overexpression showed the same results (29.38 vs 51.22 %, p < 0.05 and 52.67 vs 41.38 %, p < 0.05, respectively) (Fig. 4a and Additional file 3: Figure S2a). On the contrary, decrease in the percentage of G1-phase cells and increase in the percentage of S-phase were observed after endogenous TLE3 in HCT15 and SW620 cells was knocked down. The percentage of S-phase cells in HCT15 and SW620 cells of TLE3 knock-down were significantly more than that in HCT15 and SW620 cells of control group (40.35 vs 50.88 %, p < 0.05 and 29.49 vs 39.88 %, p < 0.05, respectively) (Fig. 4b and Additional file 3: Figure S2b). Moreover, TLE3 overexpressing significantly increased the percentage of apoptotic cells in SW480 and Ls174t cells, whereas knockdown of TLE3 in SW620 and HCT15 cells decreased the number of apoptotic cells (Fig. 4c and d, Additional file 3: Figure S2c and d). Taken together, these results demonstrate that TLE3 inhibits cell cycle progression and promotes cell death in CRC cells.Fig. 4


TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways
TLE3 inhibited cell cycle progression of human CRC cell. a, b Representative figures depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. c, d Flow cytometry of annexin V and PI-labelled CRC cells indicating apoptosis
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037636&req=5

Fig4: TLE3 inhibited cell cycle progression of human CRC cell. a, b Representative figures depicting cell cycle profiles of indicated cells. Cells were stained with PI and analyzed by flow cytometry. c, d Flow cytometry of annexin V and PI-labelled CRC cells indicating apoptosis
Mentions: To explore the possible mechanism by which TLE3 regulates the proliferation of human CRC cells, we analyzed TLE3 RNA expression levels based on TCGA COAD RNA Seq dataset and cycling gene signatures from the online Gene Set Database of the gene set enrichment analysis (GSEA) [34]. We observed that TLE3 expression was negatively correlated with genes related to cell cycle and G1-S transition (Additional file 2: Figure S1a and b). Furthermore, flow cytometry was performed to measure the distribution of cell cycle phases. Compared with control cells, the percentage of G1-phase cells increased and S-phase decreased significantly in the SW480 cells with TLE3 overexpressing (27.14 vs 41.55 %, p < 0.05, and 49.47 vs 34.02 %, p < 0.05, respectively) (Fig. 4a and Additional file 3: Figure S2a). Another cell line Ls174t of TLE3 overexpression showed the same results (29.38 vs 51.22 %, p < 0.05 and 52.67 vs 41.38 %, p < 0.05, respectively) (Fig. 4a and Additional file 3: Figure S2a). On the contrary, decrease in the percentage of G1-phase cells and increase in the percentage of S-phase were observed after endogenous TLE3 in HCT15 and SW620 cells was knocked down. The percentage of S-phase cells in HCT15 and SW620 cells of TLE3 knock-down were significantly more than that in HCT15 and SW620 cells of control group (40.35 vs 50.88 %, p < 0.05 and 29.49 vs 39.88 %, p < 0.05, respectively) (Fig. 4b and Additional file 3: Figure S2b). Moreover, TLE3 overexpressing significantly increased the percentage of apoptotic cells in SW480 and Ls174t cells, whereas knockdown of TLE3 in SW620 and HCT15 cells decreased the number of apoptotic cells (Fig. 4c and d, Additional file 3: Figure S2c and d). Taken together, these results demonstrate that TLE3 inhibits cell cycle progression and promotes cell death in CRC cells.Fig. 4

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