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Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2.

Li D, Mei H, Pu J, Xiang X, Zhao X, Qu H, Huang K, Zheng L, Tong Q - Mol. Cancer (2015)

Bottom Line: Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH.Patients with high ITLN1 or NDRG2 expression had greater survival probability.These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. d.li26@hotmail.com.

ABSTRACT

Background: Recent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. However, its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown.

Methods: Human neuroblastoma cell lines were treated with recombinant ITLN1 protein or stably transfected with ITLN1 expression and short hairpin RNA vectors. Gene expression and signaling pathway were detected by western blot and real-time quantitative RT-PCR. Gene promoter activity and transcription factor binding were detected by luciferase reporter and chromatin immunoprecipitation assays. Growth and aggressiveness of tumor cells were measured by MTT colorimetry, colony formation, scratch assay, matrigel invasion assay, and nude mice model.

Results: Mining of public microarray databases revealed that N-myc downstream regulated gene 2 (NDRG2) was significantly correlated with ITLN1 in NB. Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH. Krüppel-like factor 4 (KLF4), a transcription factor crucial for NDRG2 expression, was up-regulated by ITLN1 in NB cells via inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling. Ectopic expression of ITLN1 suppressed the growth, invasion and metastasis of NB cells in vitro and in vivo. Conversely, knockdown of ITLN1 promoted the growth, invasion, and metastasis of NB cells. In addition, rescue experiments in ITLN1 over-expressed or silenced NB cells showed that restoration of NDRG2 expression prevented the tumor cells from ITLN1-mediated changes in these biological features. In clinical NB tissues, ITLN1 was down-regulated and positively correlated with NDRG2 expression. Patients with high ITLN1 or NDRG2 expression had greater survival probability.

Conclusions: These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

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Related in: MedlinePlus

ITLN1 attenuates the growth and metastasis of NB cellsin vivo. (A and D) Tumor growth curve of SH-SY5Y cells (1 × 106) stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1 in athymic nude mice (n = 5 for each group), after hypodermic injection for 4 weeks. (B and E) Representation (top) and quantification (bottom) of xenograft tumors formed by hypodermic injection of SH-SY5Y cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. (C and F) Representation (top, arrowhead) and quantification (bottom) of lung metastasis after injection of SH-SY5Y cells (0.4 × 106) stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1 into the tail vein of athymic nude mice (n = 5 for each group). **P < 0.001 vs. mock or sh-Scb.
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Fig5: ITLN1 attenuates the growth and metastasis of NB cellsin vivo. (A and D) Tumor growth curve of SH-SY5Y cells (1 × 106) stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1 in athymic nude mice (n = 5 for each group), after hypodermic injection for 4 weeks. (B and E) Representation (top) and quantification (bottom) of xenograft tumors formed by hypodermic injection of SH-SY5Y cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. (C and F) Representation (top, arrowhead) and quantification (bottom) of lung metastasis after injection of SH-SY5Y cells (0.4 × 106) stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1 into the tail vein of athymic nude mice (n = 5 for each group). **P < 0.001 vs. mock or sh-Scb.

Mentions: We next investigated the efficacy of ITLN1 against tumor growth and metastasis in vivo. Stable transfection of ITLN1 into SH-SY5Y cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice, when compared to those stably transfected with empty vector (mock) (Figure 5A and B). In the experimental metastasis studies, SH-SY5Y cells stably transfected with ITLN1 established statistically fewer lung metastatic colonies than mock group (Figure 5C). On the other hand, stable knockdown of ITLN1 in SH-SY5Y cells resulted in increased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice (Figure 5D and E), and more lung metastatic colonies (Figure 5F), than those stably transfected with sh-Scb. These results are consistent with the findings that ITLN1 suppresses the growth, migration, and invasion of NB cells in vitro. Accordingly, identification of NDRG2 as the target gene of ITLN1 may explain, at least in part, why over-expression of ITLN1 suppresses the aggressiveness of NB.Figure 5


Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2.

Li D, Mei H, Pu J, Xiang X, Zhao X, Qu H, Huang K, Zheng L, Tong Q - Mol. Cancer (2015)

ITLN1 attenuates the growth and metastasis of NB cellsin vivo. (A and D) Tumor growth curve of SH-SY5Y cells (1 × 106) stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1 in athymic nude mice (n = 5 for each group), after hypodermic injection for 4 weeks. (B and E) Representation (top) and quantification (bottom) of xenograft tumors formed by hypodermic injection of SH-SY5Y cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. (C and F) Representation (top, arrowhead) and quantification (bottom) of lung metastasis after injection of SH-SY5Y cells (0.4 × 106) stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1 into the tail vein of athymic nude mice (n = 5 for each group). **P < 0.001 vs. mock or sh-Scb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359454&req=5

Fig5: ITLN1 attenuates the growth and metastasis of NB cellsin vivo. (A and D) Tumor growth curve of SH-SY5Y cells (1 × 106) stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1 in athymic nude mice (n = 5 for each group), after hypodermic injection for 4 weeks. (B and E) Representation (top) and quantification (bottom) of xenograft tumors formed by hypodermic injection of SH-SY5Y cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. (C and F) Representation (top, arrowhead) and quantification (bottom) of lung metastasis after injection of SH-SY5Y cells (0.4 × 106) stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1 into the tail vein of athymic nude mice (n = 5 for each group). **P < 0.001 vs. mock or sh-Scb.
Mentions: We next investigated the efficacy of ITLN1 against tumor growth and metastasis in vivo. Stable transfection of ITLN1 into SH-SY5Y cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice, when compared to those stably transfected with empty vector (mock) (Figure 5A and B). In the experimental metastasis studies, SH-SY5Y cells stably transfected with ITLN1 established statistically fewer lung metastatic colonies than mock group (Figure 5C). On the other hand, stable knockdown of ITLN1 in SH-SY5Y cells resulted in increased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice (Figure 5D and E), and more lung metastatic colonies (Figure 5F), than those stably transfected with sh-Scb. These results are consistent with the findings that ITLN1 suppresses the growth, migration, and invasion of NB cells in vitro. Accordingly, identification of NDRG2 as the target gene of ITLN1 may explain, at least in part, why over-expression of ITLN1 suppresses the aggressiveness of NB.Figure 5

Bottom Line: Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH.Patients with high ITLN1 or NDRG2 expression had greater survival probability.These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. d.li26@hotmail.com.

ABSTRACT

Background: Recent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. However, its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown.

Methods: Human neuroblastoma cell lines were treated with recombinant ITLN1 protein or stably transfected with ITLN1 expression and short hairpin RNA vectors. Gene expression and signaling pathway were detected by western blot and real-time quantitative RT-PCR. Gene promoter activity and transcription factor binding were detected by luciferase reporter and chromatin immunoprecipitation assays. Growth and aggressiveness of tumor cells were measured by MTT colorimetry, colony formation, scratch assay, matrigel invasion assay, and nude mice model.

Results: Mining of public microarray databases revealed that N-myc downstream regulated gene 2 (NDRG2) was significantly correlated with ITLN1 in NB. Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH. Krüppel-like factor 4 (KLF4), a transcription factor crucial for NDRG2 expression, was up-regulated by ITLN1 in NB cells via inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling. Ectopic expression of ITLN1 suppressed the growth, invasion and metastasis of NB cells in vitro and in vivo. Conversely, knockdown of ITLN1 promoted the growth, invasion, and metastasis of NB cells. In addition, rescue experiments in ITLN1 over-expressed or silenced NB cells showed that restoration of NDRG2 expression prevented the tumor cells from ITLN1-mediated changes in these biological features. In clinical NB tissues, ITLN1 was down-regulated and positively correlated with NDRG2 expression. Patients with high ITLN1 or NDRG2 expression had greater survival probability.

Conclusions: These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.

Show MeSH
Related in: MedlinePlus