Galectin‑1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

Abstract

The present study aimed to investigate the potential molecular mechanisms by which galectin‑1 (Gal‑1) and glucose‑regulated protein 78 (GRP78) influence the development of malignant gastric cancer (GC). Immunohistochemistry and western blotting were used to map the expression and location of the Gal‑1 gene in the 80 paraffin‑embedded GC samples, 16 fresh samples and surrounding tissues. Gal‑1 was overexpressed and knocked down using lentiviral vectors in the human GC cell lines HGC‑27 and AGS. Through the use of the Cell Counting Kit‑8 assay, clone formation assay, wound healing assay, invasion assay and tumor xenograft, the possible biological roles of Gal‑1 were further evaluated. The downstream interacting proteins were predicted by the BioGRID database, and GRP78 was chosen for further investigation. Immunofluorescence labeling and Co‑IP were used to confirm the connection. The statistical tests utilized were the two‑tailed paired Student's t‑test, χ² test, Kaplan‑Meier and Cox regression analysis, and Spearman's rank correlation coefficients. In GC, Gal‑1 is extensively expressed and has the potential to interact with GRP78. Poor prognosis is linked to high levels of GRP78 and Gal‑1 expression in patients with GC. According to the functional study, Gal‑1 knockdown prevented cells from thriving and pushed Gal‑1 expression, which aided in the proliferation, migration and invasion of GC. Gal‑1 overexpression additionally aided the development of subcutaneous xenograft tumors. The mechanistic investigation proved that GRP78 and Gal‑1 interacted, accelerating the course of GC. Gal‑1 silencing had an inhibitory effect on the proliferation of HGC‑27 cells that was removed by ectopic GRP78 expression, whereas the stimulating effects of Gal‑1 overexpression in AGS cells were inhibited by GRP78 knockdown. In conclusion, Gal‑1 interacts with GRP78 to facilitate the advancement of GC. The Gal‑1/GRP78 axis is supported by the functional data of the present study as a possible GC treatment target.

Keywords: galectin‑1; gastric cancer; glucose‑regulated protein 78; prognosis; progression.

Figure 1

Gal-1 is upregulated in gastric cancer and is associated with a poor prognosis of gastric cancer. (A) Gal-1 expression in GC (n=408) and normal (n=211) samples was evaluated using the online tool GEPIA. (B and C) The box plot of expression value of Gal-1 in gastric cancer. The X-axis represents normal (left) and cancer group (right), Y-axis represents mRNA expression in log2 median/mean-centered intensity. (D) Western blot analysis results of Gal-1 in 16 pairs of normal gastric tissue and gastric cancer tissue. (E) Gal-1 expression of 16 fresh paired GC and adjacent tissue samples were determined by western blotting. (F) Immunohistochemical test results of galectin-1 in adjacent gastric tissue and gastric cancer tissue. Representative images are shown (magnification, ×200). (G) Qualification of gal-1 staining in TMAs in panel F. The graph depicts the total score, the multiplication product of staining intensity and the percentage of stained cells. (H) Survival analysis of gastric cancer patients with different expression levels of galectin-1. (I) The expression level of gal-1 in normal gastric epithelial cells GES-1 and gastric cancer cells. ^*P<0.05, ^**P<0.01 and ^***P<0.001. GC, gastric cancer; Gal-1, galectin-1.

Figure 2

(A) The cell fluorescence expression was observed after 72 h-transfection (magnification, ×200). (B) The knockdown efficiencies of gal-1 in the shGal-1 group in HGC-27 cells and the OE efficiencies of gal-1 in the OE-Gal-1 group in AGS cells were detected by reverse transcription-quantitative PCR. (C) Western blot analysis was used to verify the constructed galectin-1 OE and galectin-1 knockdown gastric cancer cell lines. (D and E) Detection of the proliferation of (D) HGC-27 cells after Gal-1 knockdown and (E) AGS cells after Gal-1 OE by Cell Counting Kit-8 assay. ^*P<0.05, ^**P<0.01 and ^***P<0.001. Gal-1, galectin-1; sh-, short hairpin; OE, overexpression.

Figure 3

Gal-1 knockdown inhibits GC cell proliferation, migration and invasion, and Gal-1 OE promotes GC cell proliferation migration and invasion. (A and B) Detection of the proliferation of (A) HGC-27 cells after Gal-1 knockdown and (B) AGS cells after Gal-1 OE by colony formation assays. (C and D) Detection of the migration of (C) HGC-27 cells after Gal-1 knockdown and (D) AGS cells after Gal-1 OE by wound healing assay. (E and F) Detection of the migration and invasion of (E) HGC-27 cells after Gal-1 knockdown and (F) AGS cells after OE by Transwell assays. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. Gal-1, galectin-1; GC, gastric cancer; sh-, short hairpin; OE, overexpression.

Figure 4

Effects of OE of Gal-1 on tumorigenesis in nude mice in vivo. (A) Xenograft models in nude mice were generated using AGS cells transfected with Ctrl (n=5) or Gal-1 lentiviral OE vector (n=5). (B) Subcutaneous tumor growth curve in nude mice. (C) Subcutaneous tumor images. (D) Detection of the tumor weight of AGS cells transfected with Gal-1 lentiviral OE vector after euthanasia. (E) Immunohistochemical detection of Gal-1 and Ki67 in xenograft tumors formed by AGS. ^**P<0.01 and ^***P<0.001. OE, overexpression; Gal-1, galectin-1.

Figure 5

GRP78 is associated with Gal-1. (A) The interaction protein network of Gal-1 was revealed by the BioGRID database (https://thebiogrid.org/). (B) GRP78 mRNA expression in GC (n=408) and average (n=211) samples were evaluated using the online tool GEPIA. (C) The expression level of Gal-1 mRNA is independent of GRP78 mRNA in The Cancer Genome Atlas database. (D and E) Gal-1 physically interacted with GRP78. The endogenous proteins from HGC-27 cells were immunoprecipitated with IgG or antibodies against Gal-1 and GRP78, followed by western blot analysis and cell lysis for input. (F) Immunofluorescence colocalization of Gal-1 and GRP78 in GC cells (scale bar, 10 µm). (G) A western blot assay was performed to detect the GRP78 protein levels in the HGC-27 cells, knocking-down Gal-1. (H) Western blot analysis was performed to detect the GRP78 protein levels in the AGS cells overexpressing Gal-1. ^*P<0.05. GRP78, glucose-regulated protein 78; Gal-1, galectin-1; GC, gastric cancer; sh-, short hairpin; OE, overexpression.

Figure 6

Tumor-promoting effect of Gal-1 is dependent on the upregulation of GRP78. (A) The knockdown and overexpression efficiency of GRP78 in AGS and HGC-27 cells was confirmed by western blot analysis. (B) Colony formation assay revealed that ectopic GRP78 expression restored the number of cell colonies in shGal-1HGC-27 cells. (C) Ectopic GRP78 expression promoted migration and invasion in shGal-1 HGC-27 cells. (D) Colony formation assay showed that lentivirus-mediated knockdown of GRP78, and ectopic Gal-1 expression inhibited cell growth of AGS cells. (E) Knockout GRP78 inhibited migration and invasion in OE-Gal-1 AGS cells. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. Gal-1, galectin-1; GRP78, glucose-regulated protein 78; sh-, short hairpin; OE, overexpression.

Figure 7

(A) The IHC assay of Gal-1 and GRP78 expression in GC tissues and corresponding adjacent normal tissues was performed, and the representative images were shown. (B) Qualification of GRP78 staining in TMAs described in panel A. The graph depicts the total score, the multiplication product of staining intensity and the percentage of stained cells. (C) Statistical analysis of the relationship between the expression level of GRP78 and Gal-1 (P-value: Spearman's correlation coefficient). (D) Kaplan-Meier overall survival curves for all 80 patients with GC stratified by high and low expression of GRP78. ^**P<0.01. Gal-1, galectin-1; GRP78, glucose-regulated protein 78; IHC, immunohistochemical; GC, gastric cancer.