Gene silencing of galectin-3 changes the biological behavior of Eca109 human esophageal cancer cells

Abstract

Galectin-3 is a multifunctional β-galactoside‑binding lectin that is involved in multiple biological functions which are upregulated in malignancies, including cell growth, adhesion, proliferation, progression and metastasis, as well as apoptosis. A previous study has confirmed the roles of galecin-3 overexpression in the biological behavior of Eca109 human esophageal cancer (EC) cells. In the present study, small interfering (si)RNA-mediated galectin-3 silencing was performed to analyze the effects of decreased galectin-3 expression on the biological behavior of EC cells. Western blot and quantitative polymerase chain reaction analyses were utilized to confirm galectin-3 knockdown at the protein and mRNA level (P<0.05 vs. siRNA-control and untransfected groups). Cell proliferation was assessed using the Cell Counting Kit-8 assay. At 72 and 96 h after transfection, the proliferation of Eca109 cells in the siRNA-Gal-3 group was decreased compared with that in the siRNA-Control and untransfected groups (P<0.001 and P=0.004, respectively). Furthermore, Transwell assays demonstrated that inhibition of galecin-3 significantly reduced the migration and invasion of Eca109 cells compared with that in the other groups (P<0.05). Finally, apoptosis of Eca109 cells was detected using Annexin V/7-amino‑actinomycin double-staining and flow cytometric analysis. Galectin-3 knockdown significantly enhanced the apoptotic rate of Eca109 cells compared with that in the siRNA-control and untreated groups (P=0.031 and P=0.047, respectively). In conclusion, following successful knockdown of galecin-3 expression in Eca109 cells, the cell proliferation, migration and invasion were reduced, while the apoptosis was enhanced, which indicates that galectin silencing may represent a therapeutic strategy for EC.

Figure 1

Protein and mRNA expression levels of galectin-3 in Eca109 human esophageal cancer cells. (A and B) Protein expression of galectin-3 in siGal3-1, siGal3-2, siRNA-Control and untreated cells was determined by western blot analysis 72 h post-transfection. A 31-kDa band representing the location of galectin-3 was visualized in four groups. Galectin-3 expression was significantly reduced in the siGal-3 groups compared with that in the in siRNA-Control and untreated groups (P<0.05). (C) mRNA expression of galectin-3 in the four groups 48 h post-transfection. Compared with the siRNA-Control and untreated groups, galectin-3 was significantly reduced in the siGal-3 groups (P<0.001). Values are expressed as the mean ± standard deviation. ^*P≤0.05. siRNA, small interfering RNA; Gal, galectin.

Figure 2

Proliferation capacity of siRNA-Gal-3- and siRNA-control-transfected as well as untreated Eca109 cells. At (A) 24 h and (B) 48 h after transfection with siRNA, no significant differences were observed among three groups (P>0.05). (C) At 72 h after transfection, the proliferation of Eca109 cells in the siRNA-Gal-3 group was lower than that in the siRNA-control group. (D) At 96 h after transfection, the siRNA-Gal-3 treated group showed a significantly decreased proliferation compared with that in the siRNA-control and untreated groups (P=0.004 and P=0.001, respectively). (E) Cell proliferation curve of Eca109 cells in the three groups. Values are expressed as the mean ± standard deviation. ^*P≤0.05. siRNA, small interfering RNA; Gal, galectin.

Figure 3

Gal-3 knockdown inhibits the invasive and migratory abilities of Eca109 cells. Microscopy images of crystal violet-stained cells in the siRNA-Gal-3, siRNA-Control and untreated groups, respectively, on the lower side of Transwell membranes (magnification, ×4.2) in (A–C) the invasion and (D–F) the migration assay. (G) Quantification of invaded cells revealed significantly decreased invasion in the siRNA-Gal-3 group compared with that in the siRNA- Control and untreated groups (P=0.002 and P=0.001, respectively). (H) Quantification of migrated cells revealed reduced migration of siRNA-Gal-3-treated Eca109 cells compared with that in the siRNA-Control and untreated groups (P=0.001 and P<0.001, respectively). Values are expressed as the mean ± standard deviation. ^*P≤0.05. siRNA, small interfering RNA; Gal, galectin.

Figure 4

Gal-3 knockdown enhances apoptosis of Eca109 cells. Flow cytometric analysis of apoptotic cells in the (A) siRNA-Gal-3, (B) siRNA-Control and (C) untreated groups. (D) The siRNA-Gal-3 group exhibited a significantly higher apoptotic rate than the siRNA-Control and the untreated group (P=0.031 and P=0.047, respectively). Values are expressed as the mean ± standard deviation. ^*P≤0.05. siRNA, small interfering RNA; Gal, galectin; 7-AAD, 7-amino-actinomycin; PE, phycoerythrin.