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. 2014 May 30;5(10):3386-98.
doi: 10.18632/oncotarget.1960.

Up-regulation of neogenin-1 Increases Cell Proliferation and Motility in Gastric Cancer

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Free PMC article

Up-regulation of neogenin-1 Increases Cell Proliferation and Motility in Gastric Cancer

Seok-Jun Kim et al. Oncotarget. .
Free PMC article

Abstract

Although elevated expression of neogenin-1 has been detected in human gastric cancer tissue, its role in gastric tumorigenesis remains unclear due to the lack of neogenin-1 studies in cancer. Therefore, we demonstrated here the function and regulatory mechanism of neogenin-1 in gastric cancer. Neogenin-1 ablation decreased proliferation and migration of gastric cancer cells, whereas its over-expression reversed these effects. Xenografted analyses using gastric cancer cells displayed statistically significant inhibition of tumor growth by neogenin-1 depletion. Interestingly, galectin-3 interacted with HSF-1 directly, which facilitated nuclear-localization and binding on neogenin-1 promoter to drive its transcription and gastric cancer cell motility. The galectin-3-increased gastric cancer cell motility was down-regulated by HSF-1 depletion. Moreover, the parallel expression patterns of galectin-3 and neogenin-1, as well as those of HSF-1 and neogenin-1, were detected in the malignant tissues of gastric cancer patients. Taken together, high-expression of neogenin-1 promotes gastric cancer proliferation and motility and its expression is regulated by HSF-1 and galectin-3 interaction. In addition, we propose further studies for neogenin-1 and its associated pathways to provide them as a proper target for gastric cancer therapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Immunohistochemical analysis of neogenin-1 expression in human gastric cancer patient tissues
The evaluation of neogenin-1 expression investigated in gastric cancers. (A) Adjacent normal tissues of gastric cancer, (B) Intestinal type adenocarcinoma gastric cancer tissue, (C) Diffuse type adenocarcinoma gastric cancer tissue, (D and E) signet ring cell carcinoma gastric cancer, on tissue microarrays by immunohistochemical method. Neogenin-1 (brown) is mostly expressed in cytoplasms of each tissue through staining intensities. (A-D) panels magnification, x 200, (E) panel magnification, x400
Figure 2
Figure 2. Effect of each ablation of neogenin-1 or netrin-1 on the proliferation and migration of six gastric cancer cells and on AGS xenografted mice
(A) Cell proliferation was checked in 6 gastric cancer cell lines (AGS, MKN-28, YCC-2, SNU-216, SNU-601, and SNU-668) after transfection with scRNAs, neogenin-1 siRNA or netrin-1 siRNA (* p<0.001). (B) Six gastric cancer cell lines were transfected with scRNAs, neogenin-1 siRNA or netrin-1 siRNA, and analyzed by migration and invasion assays, with the results presented as histograms (* p<0.04). All experiments were performed in triplicate. (C) The effect of neogenin-1 on tumor growth of xenografts in nude mice. 50ul of AGS SQ cells (1 × 106) with Matrigel were implanted into Balb/c-nude mice to form subcutaneous xenografts. Tumor volumes were measured at different time points and are presented in photographs taken at day 28 and in a graph (D). Data are presented as the mean and standard error of the mean (SEM). Unpaired student's t-test was used for the comparison between the two groups (*, ** p<0.002).
Figure 3
Figure 3. Effect of transient transfection of neogenin-1 in SNU-668 gastric cancer cells on the proliferation, migration and invasion
(A-C) SNU-668 cells were transfected with pcDNA3.0 control vector and pcDNA3.0-neogenin-1 expression vector for 48 h. (A) Western blot analysis of neogenin-1 protein expression and crystal violet staining of cells, (B) Cell proliferation measured by WST assay (* p<0.001), (C) Cell migration assay and cell invasion assay (* p<0.0001), were performed as described in the methods. (D) Expression levels of neogenin-1 and ROCK1-1 were detected by RT-PCR and Western blot analysis in SNU-668 cells, which were infected with lentivirus-containing LacZ or neogenin-1 full-length, and then were transfected with scRNA or ROCK1 siRNA. β-actin was used as a loading control. (E) The cells were analyzed with cell migration and invasion assays, and data are presented as histograms (*, ** p<0.001). All experiments were performed in triplicate.
Figure 4
Figure 4. Effect of galectin-3 depletion on the expression of neogenin-1 in human gastric cancer cells
(A) mRNA and protein levels of galectin-3 and neogenin-1 after transfection of AGS cells with scrambled siRNA (scRNA), galectin-3 siRNA, or neogenin-1 siRNA. β-actin was used as a loading control. (B) Expression levels of galectin-3 and neogenin-1 in SNU-638 cells as determined by western blotting after transfection with pcDNA3.1/NT-GFP-galectin-3 and vector control of pcDNA3.1/NT-GFP. (C-D) After transfection of AGS cells with scrambled siRNA (scRNA), galectin-3 siRNA, or neogenin-1 siRNA, the migration and invasion assay were performed as described in “Methods” and quantification of (C) cell migration (*, ** p<0.0001) and (D) cell invasion are presented as a histogram (*, ** p<0.0001). All experiments were performed in triplicate.
Figure 5
Figure 5. Transcriptional regulation of neogenin-1 through interaction with galectin-3 and HSF-1 transcriptional factor
(A) The proximal HSF-1 binding site is located between -1478 and -1470 from the initiation codon of NEO1 gene. (B) Chromatin immunoprecipitation assay was performed as described in “Methods.” Total genomic DNA was used in the input lane as a control for the PCR assay. (C) Luciferase activity of HSF-1 in AGS cells treated with galectin-3 siRNA or HSF-1 siRNA. A luciferase assay was performed using a HSF-1 binding site luciferase vector transfection with galectin-3 siRNA or HSF-1 siRNA (*, ** p<0.001). The experiments were performed in triplicate. (D) The detection of the interaction between galectin-3 and HSF-1 by immunoprecipitation was performed as described in “Methods,” and then galectin-3 and HSF-1 were detected by western blot analysis. Whole-cell lysate (WCL) was used as a loading control. (E) Protein levels of galectin-3 and HSF-1 were detected in the nuclear and cytosol fractions of AGS cells which were transfected with galectin-3 or HSF-1 siRNA. Lamin A/C was used as a control for the nuclear fraction.
Figure 6
Figure 6. Effect of HSF-1 depletion on the expression of neogenin-1 and gastric cancer cell motility
(A) Detection of mRNA and protein levels of galectin-3, HSF-1, HSP70, and neogenin-1 after transfection of AGS cells with scRNA, galectin-3 siRNA and HSF-1 siRNA. β-actin was used as a loading control. (B) After transfection of AGS cells with scrambled siRNA (scRNA), or HSF-1 siRNA, the migration assay were performed and quantification of cell migration was presented as a histogram (*, ** p<0.001). (C) Expression levels of galectin-3, HSF-1, HSP70 and neogenin-1 were detected by RT-PCR and Western blotting analysis in SNU-638 cells, which were infected with lentivirus-containing LacZ or galectin-3 full-length, and then followed by transfection with HSF-1 siRNA or scRNA. β-actin was used as a loading control. (D) The cells were analyzed by migration assay and invasion assay, and data are presented as histograms (*, ** p<0.001). All experiments were performed in triplicate.
Figure 7
Figure 7. Positive association between galectin-3 and neogenin-1 expression, and HSF-1 and neogein1 expression in malignant and normal tissues from gastric cancer patients
(A) mRNA expression levels of galectin-3 and neogenin-1 from malignant tissues of 20 gastric cancer patients are presented in a histogram. (B) Expression levels of galectin-3 and neogenin-1 were calculated using ImageJ in normal and tumor tissues of 20 gastric cancer patients. Those p values were calculated using student T-test. (C) Protein expression of HSF-1 and neogenin-1 in the malignant tissues of gastric cancer patients were shown by immunohistochemical staining (brown) with hematoxylin and eosin (H&E) and detected by inverted light microscope. Magnification; X200 (up) and X400 (down).

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