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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

Involvement of KLF4 in ITLN1-mediated up-regulation of NDRG2. (A) Scheme of the potential binding site of KLF4 within NDRG2 promoter. (B) Luciferase reporter assay showing the activity of NDRG2 promoter and its mutant in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (C) ChIP and qPCR assays showing the binding of KLF4 on −133/+55 region of NDRG2 promoter in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and co-transfected with sh-KLF4 or KLF4. (D) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)], KLF4, and NDRG2 (in lysate) in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (E) The transcript levels of ITLN1, KLF4, and NDRG2 in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4 as detected by real-time quantitative RT-PCR. *P < 0.01 vs. mock or sh-Scb.
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Fig2: Involvement of KLF4 in ITLN1-mediated up-regulation of NDRG2. (A) Scheme of the potential binding site of KLF4 within NDRG2 promoter. (B) Luciferase reporter assay showing the activity of NDRG2 promoter and its mutant in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (C) ChIP and qPCR assays showing the binding of KLF4 on −133/+55 region of NDRG2 promoter in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and co-transfected with sh-KLF4 or KLF4. (D) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)], KLF4, and NDRG2 (in lysate) in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (E) The transcript levels of ITLN1, KLF4, and NDRG2 in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4 as detected by real-time quantitative RT-PCR. *P < 0.01 vs. mock or sh-Scb.

Mentions: To investigate the mechanisms underlying ITLN1-mediated up-regulation of NDRG2, we analyzed the transcription factor binding sites within NDRG2 promoter, and noted one potential binding site of transcription factor KLF4, locating at bases 7–18 upstream the TSS (Figure 2A). The KLF4 levels were correlated with NDRG2 expression (correlation coefficient R = 0.441, P < 0.001) and greater survival probability (P = 0.027) in 88 NB cases derived from R2 microarray analysis and visualization platform (Additional file 4: Figure S4). Dual-luciferase assay indicated that ectopic expression or knockdown of KLF4 increased and decreased the promoter activity of NDRG2 in NB cells, and the ITLN1-facilitated NDRG2 promoter activity was abolished by mutation of KLF4 binding site (Figure 2B). In addition, chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) indicated that over-expression or knockdown of ITLN1 increased or decreased the binding of KLF4 on −133/+55 region of NDRG2 promoter in SH-SY5Y and SK-N-SH cells, which was rescued by knockdown or ectopic expression of KLF4, respectively (Figure 2C). Moreover, western blot and real-time quantitative RT-PCR indicated that ITLN1 up-regulated the protein levels, but not the transcript levels, of KLF4 in NB cells (Figure 2D and E), indicating that ITLN1 may facilitate the expression of KLF4 at the translational level. Knockdown or ectopic expression of KLF4 into NB cells prevented the NB cells from ITLN1-mediated changes in NDRG2 expression (Figure 2D and E). These results indicate that KLF4 facilitates the transcription of NDRG2, and plays a crucial role in ITLN1-induced up-regulation of NDRG2 in NB cells.Figure 2


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)

Involvement of KLF4 in ITLN1-mediated up-regulation of NDRG2. (A) Scheme of the potential binding site of KLF4 within NDRG2 promoter. (B) Luciferase reporter assay showing the activity of NDRG2 promoter and its mutant in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (C) ChIP and qPCR assays showing the binding of KLF4 on −133/+55 region of NDRG2 promoter in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and co-transfected with sh-KLF4 or KLF4. (D) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)], KLF4, and NDRG2 (in lysate) in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (E) The transcript levels of ITLN1, KLF4, and NDRG2 in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4 as detected by real-time quantitative RT-PCR. *P < 0.01 vs. mock or sh-Scb.
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Related In: Results  -  Collection

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Fig2: Involvement of KLF4 in ITLN1-mediated up-regulation of NDRG2. (A) Scheme of the potential binding site of KLF4 within NDRG2 promoter. (B) Luciferase reporter assay showing the activity of NDRG2 promoter and its mutant in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (C) ChIP and qPCR assays showing the binding of KLF4 on −133/+55 region of NDRG2 promoter in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and co-transfected with sh-KLF4 or KLF4. (D) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)], KLF4, and NDRG2 (in lysate) in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4. (E) The transcript levels of ITLN1, KLF4, and NDRG2 in NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-KLF4 or KLF4 as detected by real-time quantitative RT-PCR. *P < 0.01 vs. mock or sh-Scb.
Mentions: To investigate the mechanisms underlying ITLN1-mediated up-regulation of NDRG2, we analyzed the transcription factor binding sites within NDRG2 promoter, and noted one potential binding site of transcription factor KLF4, locating at bases 7–18 upstream the TSS (Figure 2A). The KLF4 levels were correlated with NDRG2 expression (correlation coefficient R = 0.441, P < 0.001) and greater survival probability (P = 0.027) in 88 NB cases derived from R2 microarray analysis and visualization platform (Additional file 4: Figure S4). Dual-luciferase assay indicated that ectopic expression or knockdown of KLF4 increased and decreased the promoter activity of NDRG2 in NB cells, and the ITLN1-facilitated NDRG2 promoter activity was abolished by mutation of KLF4 binding site (Figure 2B). In addition, chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) indicated that over-expression or knockdown of ITLN1 increased or decreased the binding of KLF4 on −133/+55 region of NDRG2 promoter in SH-SY5Y and SK-N-SH cells, which was rescued by knockdown or ectopic expression of KLF4, respectively (Figure 2C). Moreover, western blot and real-time quantitative RT-PCR indicated that ITLN1 up-regulated the protein levels, but not the transcript levels, of KLF4 in NB cells (Figure 2D and E), indicating that ITLN1 may facilitate the expression of KLF4 at the translational level. Knockdown or ectopic expression of KLF4 into NB cells prevented the NB cells from ITLN1-mediated changes in NDRG2 expression (Figure 2D and E). These results indicate that KLF4 facilitates the transcription of NDRG2, and plays a crucial role in ITLN1-induced up-regulation of NDRG2 in NB cells.Figure 2

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