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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 suppresses the growth, migration, and invasion of NB cellsin vitrothrough up-regulating NDRG2. (A and E) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)] and NDRG2 (in lysate) in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (B and F) Quantification of colony formation assay showing the growth potential of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (Cand G) Migration of NB cells upon transfection with mock, ITLN1, NDRG2, sh-Scb, sh-ITLN1, or sh-NDRG2 depicted by scratch assay after 24 hrs. (D and H) Representation (top) and quantification (bottom) of matrigel invasion assay showing the in vitro invasion of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. *P < 0.01 vs. mock or sh-Scb.
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Fig4: ITLN1 suppresses the growth, migration, and invasion of NB cellsin vitrothrough up-regulating NDRG2. (A and E) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)] and NDRG2 (in lysate) in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (B and F) Quantification of colony formation assay showing the growth potential of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (Cand G) Migration of NB cells upon transfection with mock, ITLN1, NDRG2, sh-Scb, sh-ITLN1, or sh-NDRG2 depicted by scratch assay after 24 hrs. (D and H) Representation (top) and quantification (bottom) of matrigel invasion assay showing the in vitro invasion of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. *P < 0.01 vs. mock or sh-Scb.

Mentions: We further investigated the effects of ITLN1 over-expression and target gene restoration on cultured NB cells. Transfection of short hairpin RNA (shRNA) targeting NDRG2 restored the ITLN1-induced up-regulation of NDRG2 in SH-SY5Y and SK-N-BE(2) cells (Figure 4A and Additional file 5: Figure S5A). In line with the results from MTT colorimetric assay (Additional file 6: Figure S6A), colony formation assay indicated that ITLN1 over-expression attenuated the growth of SH-SY5Y and SK-N-BE(2) cells, when compared to those stably transfected with empty vector (mock) (Figure 4B and Additional file 6: Figure S6B). In scratch assay, ITLN1 over-expression attenuated the migration capabilities of SH-SY5Y and SK-N-BE(2) cells (Figure 4C and Additional file 6: Figure S6C). Transwell analysis showed that NB cells stably transfected with ITLN1 presented an impaired invasion capacity than mock cells (Figure 4D). In addition, restoration of NDRG2 expression rescued the NB cells from their changes in these phenotypes induced by stable over-expression of ITLN1 (Figure 4B, C, and D, Additional file 6: Figure S6A, B, and C). These results reveal the tumor suppressive roles of ITLN1, and indicate that up-regulation of NDRG2 is involved in the ITLN1-inhibited aggressiveness of NB cells.Figure 4


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 suppresses the growth, migration, and invasion of NB cellsin vitrothrough up-regulating NDRG2. (A and E) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)] and NDRG2 (in lysate) in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (B and F) Quantification of colony formation assay showing the growth potential of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (Cand G) Migration of NB cells upon transfection with mock, ITLN1, NDRG2, sh-Scb, sh-ITLN1, or sh-NDRG2 depicted by scratch assay after 24 hrs. (D and H) Representation (top) and quantification (bottom) of matrigel invasion assay showing the in vitro invasion of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. *P < 0.01 vs. mock or sh-Scb.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: ITLN1 suppresses the growth, migration, and invasion of NB cellsin vitrothrough up-regulating NDRG2. (A and E) Western blot showing the protein levels of ITLN1 [in culture supernatant (s)] and NDRG2 (in lysate) in NB cells stably transfected with empty vector (mock), ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (B and F) Quantification of colony formation assay showing the growth potential of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. (Cand G) Migration of NB cells upon transfection with mock, ITLN1, NDRG2, sh-Scb, sh-ITLN1, or sh-NDRG2 depicted by scratch assay after 24 hrs. (D and H) Representation (top) and quantification (bottom) of matrigel invasion assay showing the in vitro invasion of NB cells stably transfected with mock, ITLN1, sh-Scb, or sh-ITLN1, and those co-transfected with sh-NDRG2 or NDRG2. *P < 0.01 vs. mock or sh-Scb.
Mentions: We further investigated the effects of ITLN1 over-expression and target gene restoration on cultured NB cells. Transfection of short hairpin RNA (shRNA) targeting NDRG2 restored the ITLN1-induced up-regulation of NDRG2 in SH-SY5Y and SK-N-BE(2) cells (Figure 4A and Additional file 5: Figure S5A). In line with the results from MTT colorimetric assay (Additional file 6: Figure S6A), colony formation assay indicated that ITLN1 over-expression attenuated the growth of SH-SY5Y and SK-N-BE(2) cells, when compared to those stably transfected with empty vector (mock) (Figure 4B and Additional file 6: Figure S6B). In scratch assay, ITLN1 over-expression attenuated the migration capabilities of SH-SY5Y and SK-N-BE(2) cells (Figure 4C and Additional file 6: Figure S6C). Transwell analysis showed that NB cells stably transfected with ITLN1 presented an impaired invasion capacity than mock cells (Figure 4D). In addition, restoration of NDRG2 expression rescued the NB cells from their changes in these phenotypes induced by stable over-expression of ITLN1 (Figure 4B, C, and D, Additional file 6: Figure S6A, B, and C). These results reveal the tumor suppressive roles of ITLN1, and indicate that up-regulation of NDRG2 is involved in the ITLN1-inhibited aggressiveness of NB cells.Figure 4

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