Homeobox B7 accelerates the cancer progression of gastric carcinoma cells by promoting epithelial-mesenchymal transition (EMT) and activating Src-FAK pathway

Abstract

Aim: To study the carcinogenetic mechanism of HOXB7 in gastric cancer (GC) remains. Methods: Two human GC cell lines - SGC7901 and SNU1 - were used for this study. SGC7901 cells were transfected with siRNA-HOXB7 (siHOXB7) to knock down HOXB7 expression, whereas, SNU1 cells were transduced with pCDNA3.1-HOXB7 to overexpress HOXB7. After transfection, cancer progression was assessed by determining cell proliferation, wound-healing process, cell cycle, apoptosis, invasion, and migration. The effect of HOXB7 on epithelial-mesenchymal transition (EMT) was measured by observing changes in F-actin cytoskeleton and evaluating the expression of EMT markers. p-Scr and p-FAK were evaluated to assess the mechanism. Results: Knockdown of HOXB7 suppressed cell proliferation, alleviated the wound-healing process, inhibited cell migration and invasion, and arrested the cell cycle while promoting cell apoptosis, suggesting the tumor-suppressive effect of siHOXB7 in human GC cells. On the contrary, HOXB7 overexpression showed a tumor-promoting effect on human GC cells. Moreover, we confirmed an inhibitory effect of siHOXB7 on the EMT process by preventing epithelial cells from acquiring a mesenchymal phenotype and downregulating mesenchymal markers (vimentin, β-catenin, N-cadherin, Twist) while upregulating epithelial markers (E-cadherin). Our data revealed that HOXB7 was associated with Src/FAK and favored the activation of the Src-FAK pathway in human GC cells. Conclusion: HOXB7 accelerated the malignancy of GC, by facilitating EMT and regulating the Scr-FAK pathway.

Keywords: Epithelial-mesenchymal transition; FAK; Homeobox B7; Src; gastric carcinoma.

Figure 1

Knockdown of HOXB7 inhibited the cancer progression of SGC7901 cells. Notes: SGC7900 cells were transfected with siRNA-NC (siNC) and siRNA-HOXB7 (siHOXB7-1, siHOXB7-2 and siHOXB7-3). (A) After 48 hours, mRNA and protein levels of HOXB7 were assessed by RT-PCR and Western blot, respectively. (B) After transfection at 0, 12, 24, 48, and 72 hours, cell proliferation was measured using CCK8. (C) The wound-healing process was measured by wound-healing assay at 0, 24, and 48 hours. (D) Cell cycle and (E) cell apoptosis were assessed by annexin V–PI staining. (F) Cell migration and (G) invasion were assessed with a transwell chamber (five visual fields randomly selected). **P<0.01 versus siNC.

Figure 2

HOXB7 overexpression promoted the cancer progression of SNU1 cells. SNU1cells were transfected with HOXB7overexpression/control vectors. Notes: (A) mRNA and protein levels of HOXB7 assessed by RT-PCR and Western blot, respectively. (B) Cell proliferation at 0, 12, 24, 48, and 72 hours measured using CCK8. (C) Wound-healing process measured by wound-healing assay at 0, 24, and 48 hours. (D) Cell cycle and (E) apoptosis, assessed by annexin V–PI staining. (F) Migration and (G) invasion assessed by transwell chamber (five visual fields randomly selected). ^#P<0.05; ^##P<0.01 versus vector.

Figure 3

Effect of HOXB7 on epithelial–mesenchymal transitio (EMT). Notes: After transfection, (A) F-actin cytoskeleton was observed using immunofluorescence (400×). (B) mRNA and (C) protein expression of EMT markers (E-cadherin, N-cadherin, vimentin, β-catenin, and Twist) in siHOXB7-2/siNC, or HOXB7/vector-transfected cells. **P<0.01 versus SGC7900 cells transfected with siNC; ^##P<0.01 versus SNU-1 cells transfected with vector.

Figure 4

HOXB7 was associated with Src and FAK and regulated their phosphorylation in human gastric cancer cells. Notes: Following coimmunoprecipitation assays with (A) anti-HOXB7 antibody, (B) anti-Src antibody, and (C) anti-FAK antibody, the presence of HOXB7, Src, or FAK in the immunoprecipitated protein complexes was detected by Western blot. (D) Protein expression of total Src and p-Src (Y416) and (E) total FAK, p-FAK (Y397), and p-FAK (Y576/577) within cells transfected with siHOXB7/siNC or HOXB7/vector. GAPDH was an equal loading control. p-Src or p-FAK was normalized to its corresponding total protein. **P<0.01 versus SGC7900 cells transfected with siNC; ^##P<0.01 versus SNU1 cells transfected with vector.

Figure 5

HOXB7 exerted carcinogenesis via Src and FAK in human gastric cancer cells. Notes: HOXB7/vector-transfected SNU1 cells were treated with PF573228 or PP2, and then (A, B) protein expression of total Src, p-Src (Y416), total FAK, p-FAK (Y397), and p-FAK (Y576/577) were assessed by Western blot, (C, E) cell migration and (D, F) invasion assessed by transwell assay, and (G, H) wound-healing process measured by wound-healing assay. ^##P<0.01 versus vector; ^$$P<0.01 versus HOXB7 overexpression.