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Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer.

Lv B, Song C, Wu L, Zhang Q, Hou D, Chen P, Yu S, Wang Z, Chu Y, Zhang J, Yang D, Liu J - Oncotarget (2015)

Bottom Line: However, it is completely unknown whether Ntn4 has roles in GC development.In addition, Ntn4 receptor, neogenin (Neo) was also found highly expressed in GC cells and mediated the Ntn4-induced cell proliferation and invasion.Moreover, Ntn4 or Neo silencing decreased the phosphorylation of Stat3, ERK, Akt and p38, indicating multi-oncogenic pathways (Jak/Stat, PI3K/Akt, and ERK/MAPK) were involved in Ntn4-induced effects on the GC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Digestive Diseases of Huashan Hospital, Fudan University, Shanghai, China.

ABSTRACT
Gastric cancer (GC) is the second most common cause of cancer-related death with limited serum biomarkers for diagnosis and prognosis. Netrin-4 (Ntn4) is a laminin-related secreted molecule found to regulate tumor progression and metastasis. However, it is completely unknown whether Ntn4 has roles in GC development. Here, we first reported Ntn4 knockdown significantly suppressed cell proliferation and motility, while overexpression or addition of exogenous Ntn4 reversed these effects. In addition, Ntn4 receptor, neogenin (Neo) was also found highly expressed in GC cells and mediated the Ntn4-induced cell proliferation and invasion. Moreover, Ntn4 or Neo silencing decreased the phosphorylation of Stat3, ERK, Akt and p38, indicating multi-oncogenic pathways (Jak/Stat, PI3K/Akt, and ERK/MAPK) were involved in Ntn4-induced effects on the GC cells. Importantly, Ntn4 level was significantly increased in 82 tumor tissues (p = 0.001) and 52 serum samples (p < 0.0001) from GC patients and positively correlated with Neo expression (p = 0.003). Ntn4 expression was negatively correlated with the survival period (p = 0.038), and positively associated with the severity of pathological stages of the tumors (p = 0.008). Taken together, Ntn4 promoted the proliferation and motility of GC cells which was mediated by its receptor Neo and through further activation of multi-oncogenic pathways. Elevated Ntn4 was detected in both tumor tissues and serum samples of GC patients and suggested a relatively poor survival, indicating Ntn4 may be used as a potential non-invasive biomarker for diagnosis and prognosis of GC.

No MeSH data available.


Related in: MedlinePlus

Overexpression of Ntn4 promoted the growth, migration and invasion of GC cellsA–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. **p < 0.01.
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Figure 3: Overexpression of Ntn4 promoted the growth, migration and invasion of GC cellsA–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. **p < 0.01.

Mentions: To further assess the role of Ntn4 in the proliferation and migration of GC cells, pcDNA3-Ntn4 or control vector (pcDNA3) was transiently transfected into AGS and MGC803 cells. Real-time RT-PCR and ELISA were performed to confirm Ntn4 gene overexpression at the mRNA level (Fig. 3A) and protein level (Fig. 3B). CCK8 assay revealed that the overexpression of Ntn4 obviously enhanced cell proliferation in both AGS and MGC803 cells (Fig. 3C–3D). Moreover, wound-healing assay showed that the gap sizes of AGS and MGC803 cells with pcDNA3-Ntn4 transfection were significantly decreased by 20% and 21%, respectively, at 24 h compared to that with pcDNA3 transfection (Fig. 3E). The invasion assay showed that Ntn4 overexpression was associated with an increase in invaded cells in the pcDNA3-Ntn4 group compared with control cells (Fig. 3F–3G). These data supported Ntn4 overexpression promoted cell growth, migration and invasion of GC cells, further confirming the role of Ntn4 in regulation of cell proliferation and motility.


Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer.

Lv B, Song C, Wu L, Zhang Q, Hou D, Chen P, Yu S, Wang Z, Chu Y, Zhang J, Yang D, Liu J - Oncotarget (2015)

Overexpression of Ntn4 promoted the growth, migration and invasion of GC cellsA–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496398&req=5

Figure 3: Overexpression of Ntn4 promoted the growth, migration and invasion of GC cellsA–B. The overexpression of Ntn4 was achieved in AGS and MGC cells by transient transfection of pcDNA3-Ntn4 for 48 hours and with pcDNA3 vector (pcDNA3) only as control. Expression of Ntn4 was measured with Q-PCR (A) and ELISA (B). C–D. The enhanced expression of Ntn4 accelerated the growth of AGS (C) and MGC803 (D) cells. The cell proliferation assay was performed with CCK8 assay at 24, 48 and 72 hours, respectively after the transfection with pcDNA3 or pcDNA3-Ntn4. E. The enhanced expression of Ntn4 boosted the wound healing of AGS and MGC803 cells. The monolayer of cells transfected with pcDNA3 or pcDNA3-Ntn4 was disrupted with a tip and photographed under microscope at 0 and 24 hours; Magnification, × 100. F–G. The enhanced expression of Ntn4 promoted the invasion of AGS (F) and MGC803 (G) cells. Cells were transfected with pcDNA3 or pcDNA3-Ntn4 for 48 hours and subjected to the invasion assay; Magnification, × 100. Mean ± SEM, n ≥ 3. **p < 0.01.
Mentions: To further assess the role of Ntn4 in the proliferation and migration of GC cells, pcDNA3-Ntn4 or control vector (pcDNA3) was transiently transfected into AGS and MGC803 cells. Real-time RT-PCR and ELISA were performed to confirm Ntn4 gene overexpression at the mRNA level (Fig. 3A) and protein level (Fig. 3B). CCK8 assay revealed that the overexpression of Ntn4 obviously enhanced cell proliferation in both AGS and MGC803 cells (Fig. 3C–3D). Moreover, wound-healing assay showed that the gap sizes of AGS and MGC803 cells with pcDNA3-Ntn4 transfection were significantly decreased by 20% and 21%, respectively, at 24 h compared to that with pcDNA3 transfection (Fig. 3E). The invasion assay showed that Ntn4 overexpression was associated with an increase in invaded cells in the pcDNA3-Ntn4 group compared with control cells (Fig. 3F–3G). These data supported Ntn4 overexpression promoted cell growth, migration and invasion of GC cells, further confirming the role of Ntn4 in regulation of cell proliferation and motility.

Bottom Line: However, it is completely unknown whether Ntn4 has roles in GC development.In addition, Ntn4 receptor, neogenin (Neo) was also found highly expressed in GC cells and mediated the Ntn4-induced cell proliferation and invasion.Moreover, Ntn4 or Neo silencing decreased the phosphorylation of Stat3, ERK, Akt and p38, indicating multi-oncogenic pathways (Jak/Stat, PI3K/Akt, and ERK/MAPK) were involved in Ntn4-induced effects on the GC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Digestive Diseases of Huashan Hospital, Fudan University, Shanghai, China.

ABSTRACT
Gastric cancer (GC) is the second most common cause of cancer-related death with limited serum biomarkers for diagnosis and prognosis. Netrin-4 (Ntn4) is a laminin-related secreted molecule found to regulate tumor progression and metastasis. However, it is completely unknown whether Ntn4 has roles in GC development. Here, we first reported Ntn4 knockdown significantly suppressed cell proliferation and motility, while overexpression or addition of exogenous Ntn4 reversed these effects. In addition, Ntn4 receptor, neogenin (Neo) was also found highly expressed in GC cells and mediated the Ntn4-induced cell proliferation and invasion. Moreover, Ntn4 or Neo silencing decreased the phosphorylation of Stat3, ERK, Akt and p38, indicating multi-oncogenic pathways (Jak/Stat, PI3K/Akt, and ERK/MAPK) were involved in Ntn4-induced effects on the GC cells. Importantly, Ntn4 level was significantly increased in 82 tumor tissues (p = 0.001) and 52 serum samples (p < 0.0001) from GC patients and positively correlated with Neo expression (p = 0.003). Ntn4 expression was negatively correlated with the survival period (p = 0.038), and positively associated with the severity of pathological stages of the tumors (p = 0.008). Taken together, Ntn4 promoted the proliferation and motility of GC cells which was mediated by its receptor Neo and through further activation of multi-oncogenic pathways. Elevated Ntn4 was detected in both tumor tissues and serum samples of GC patients and suggested a relatively poor survival, indicating Ntn4 may be used as a potential non-invasive biomarker for diagnosis and prognosis of GC.

No MeSH data available.


Related in: MedlinePlus