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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 facilitates the NDRG2 expression in NB cells. (A) Western blot showing the expression of NDRG2, VEGF, and MMP-9 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml, for 24 hrs) and SK-N-BE(2) (1 μg/ml, for 24 and 36 hrs) cells. (B) Western blot showing the protein levels of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in NB cells stably transfected with empty vector (mock) or ITLN1. (C) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with mock or ITLN1 as measured by real-time quantitative RT-PCR. (D) Western blot showing the expression of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in SH-SY5Y and SK-N-SH cells stably transfected with sh-Scb or sh-ITLN1. (E) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with sh-Scb or sh-ITLN1 as detected by real-time quantitative RT-PCR. (F) Luciferase reporter assay showing the activity of different NDRG2 promoter fragments in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. *P < 0.01 vs. mock or sh-Scb.
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Fig1: ITLN1 facilitates the NDRG2 expression in NB cells. (A) Western blot showing the expression of NDRG2, VEGF, and MMP-9 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml, for 24 hrs) and SK-N-BE(2) (1 μg/ml, for 24 and 36 hrs) cells. (B) Western blot showing the protein levels of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in NB cells stably transfected with empty vector (mock) or ITLN1. (C) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with mock or ITLN1 as measured by real-time quantitative RT-PCR. (D) Western blot showing the expression of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in SH-SY5Y and SK-N-SH cells stably transfected with sh-Scb or sh-ITLN1. (E) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with sh-Scb or sh-ITLN1 as detected by real-time quantitative RT-PCR. (F) Luciferase reporter assay showing the activity of different NDRG2 promoter fragments in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. *P < 0.01 vs. mock or sh-Scb.

Mentions: To address the hypothesis that ITLN1 might influence the NDRG2 expression in NB, recombinant ITLN1 protein was administrated into cultured NB cell lines SH-SY5Y and SK-N-BE(2). As shown in Figure 1A, either low dose (1 μg/ml) or high dose (2 μg/ml) of recombinant ITLN1 protein markedly induced the expression of NDRG2 in NB cells at 24 and 36 hrs post-administration. In addition, ITLN1 vector was stably transfected into SH-SY5Y and SK-N-BE(2) cells, resulting in enhanced ITLN1 expression and secretion into culture supernatant and increased NDRG2 expression levels, than those stably transfected with empty vector (mock) (Figure 1B and C). In addition, the expression of vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9), downstream target genes of NDRG2 [17,18], was also decreased in NB cells treated with recombinant ITLN1 protein or stably transfected with ITLN1 (Figure 1A, B and C). Since over-expression of NDRG2 suppressed the expression of VEGF and MMP-9 in NB cells (Additional file 2: Figure S2A), and knockdown of NDRG2 rescued ITLN1-induced down-regulation of VEGF and MMP-9 (Additional file 2: Figure S2B), we believed that ITLN1 regulated the expression of VEGF and MMP-9 through modulating NDRG2. On the other hand, stable transfection of sh-ITLN1 into SH-SY5Y and SK-N-SH cells resulted in obviously reduced expression and secretion of ITLN1, decreased NDRG2 levels, and increased expression of VEGF and MMP-9 than those of scramble short hairpin RNA (sh-Scb)-transfected cells (Figure 1D and E). In addition, stable over-expression or knockdown of ITLN1 resulted in increased and decreased NDRG2 promoter activity in NB cells, respectively, especially at −395/+192 bp region relative to the transcription start site (TSS) (Figure 1 F). In contrast, the expression of other potential target genes analyzed by R2: microarray analysis and visualization platform, including CCT3, DCUN1D5, ENO1, MACF1, and PPM1G, was not affected by ITLN1 in NB cells (Additional file 3: Figure S3). Overall, these results demonstrate that ITLN1 considerably facilitates the transcription of NDRG2 in NB cells.Figure 1


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 facilitates the NDRG2 expression in NB cells. (A) Western blot showing the expression of NDRG2, VEGF, and MMP-9 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml, for 24 hrs) and SK-N-BE(2) (1 μg/ml, for 24 and 36 hrs) cells. (B) Western blot showing the protein levels of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in NB cells stably transfected with empty vector (mock) or ITLN1. (C) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with mock or ITLN1 as measured by real-time quantitative RT-PCR. (D) Western blot showing the expression of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in SH-SY5Y and SK-N-SH cells stably transfected with sh-Scb or sh-ITLN1. (E) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with sh-Scb or sh-ITLN1 as detected by real-time quantitative RT-PCR. (F) Luciferase reporter assay showing the activity of different NDRG2 promoter fragments in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. *P < 0.01 vs. mock or sh-Scb.
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Related In: Results  -  Collection

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Fig1: ITLN1 facilitates the NDRG2 expression in NB cells. (A) Western blot showing the expression of NDRG2, VEGF, and MMP-9 in solvent (mock)- or recombinant ITLN1-treated SH-SY5Y (1 and 2 μg/ml, for 24 hrs) and SK-N-BE(2) (1 μg/ml, for 24 and 36 hrs) cells. (B) Western blot showing the protein levels of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in NB cells stably transfected with empty vector (mock) or ITLN1. (C) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with mock or ITLN1 as measured by real-time quantitative RT-PCR. (D) Western blot showing the expression of ITLN1 [in culture supernatant (s) and lysate], NDRG2, VEGF, and MMP-9 (in lysate) in SH-SY5Y and SK-N-SH cells stably transfected with sh-Scb or sh-ITLN1. (E) The transcript levels of ITLN1, NDRG2, VEGF, and MMP-9 in NB cells stably transfected with sh-Scb or sh-ITLN1 as detected by real-time quantitative RT-PCR. (F) Luciferase reporter assay showing the activity of different NDRG2 promoter fragments in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1. *P < 0.01 vs. mock or sh-Scb.
Mentions: To address the hypothesis that ITLN1 might influence the NDRG2 expression in NB, recombinant ITLN1 protein was administrated into cultured NB cell lines SH-SY5Y and SK-N-BE(2). As shown in Figure 1A, either low dose (1 μg/ml) or high dose (2 μg/ml) of recombinant ITLN1 protein markedly induced the expression of NDRG2 in NB cells at 24 and 36 hrs post-administration. In addition, ITLN1 vector was stably transfected into SH-SY5Y and SK-N-BE(2) cells, resulting in enhanced ITLN1 expression and secretion into culture supernatant and increased NDRG2 expression levels, than those stably transfected with empty vector (mock) (Figure 1B and C). In addition, the expression of vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9), downstream target genes of NDRG2 [17,18], was also decreased in NB cells treated with recombinant ITLN1 protein or stably transfected with ITLN1 (Figure 1A, B and C). Since over-expression of NDRG2 suppressed the expression of VEGF and MMP-9 in NB cells (Additional file 2: Figure S2A), and knockdown of NDRG2 rescued ITLN1-induced down-regulation of VEGF and MMP-9 (Additional file 2: Figure S2B), we believed that ITLN1 regulated the expression of VEGF and MMP-9 through modulating NDRG2. On the other hand, stable transfection of sh-ITLN1 into SH-SY5Y and SK-N-SH cells resulted in obviously reduced expression and secretion of ITLN1, decreased NDRG2 levels, and increased expression of VEGF and MMP-9 than those of scramble short hairpin RNA (sh-Scb)-transfected cells (Figure 1D and E). In addition, stable over-expression or knockdown of ITLN1 resulted in increased and decreased NDRG2 promoter activity in NB cells, respectively, especially at −395/+192 bp region relative to the transcription start site (TSS) (Figure 1 F). In contrast, the expression of other potential target genes analyzed by R2: microarray analysis and visualization platform, including CCT3, DCUN1D5, ENO1, MACF1, and PPM1G, was not affected by ITLN1 in NB cells (Additional file 3: Figure S3). Overall, these results demonstrate that ITLN1 considerably facilitates the transcription of NDRG2 in NB cells.Figure 1

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