Ovostatin 2 knockdown significantly inhibits the growth, migration, and tumorigenicity of cutaneous malignant melanoma cells

Abstract

Background: We previously identified ovostatin 2 (OVOS2) as a new candidate gene for cutaneous malignant melanoma (CMM) in a Chinese population. In this study, we aimed to investigate the exact role of OVOS2 in cell proliferation, invasion, and tumorigenesis of melanoma A375 cells.

Methods: The downregulation of OVOS2 expression was performed using lentiviral vectors with specific shRNA. The effects of OVOS2 expression on cell proliferation, cell cycle, cell migration, cell invasion, and potential of tumorigenesis were further investigated.

Results: The downregulation of OVOS2 significantly suppressed the proliferation of A375 cells and led to a G2/M phase block. The transwell cell migration assay showed that the reduced expression of OVOS2 also significantly inhibited the transmigration of A375 cells. The western blot results showed downregulated expression of p-FAK, p-AKT, and p-ERK. This was accompanied by the upregulated epithelial phenotypes E-cadherin and β-catenin, and downregulated expression of mesenchymal phenotype N-cadherin after OVOS2 knockdown. The transplantation tumor experiment in BALB/C nude mouse showed that after an observation period of 32 days, the growth speed and weight of the transplanted tumors were significantly suppressed in the BALB/c nude mice subcutaneously injected with OVOS2 knocked-down A375 cells.

Conclusion: The inhibition of OVOS2 had significant suppressive effects on the proliferation, motility, and migration capabilities of A375 cells, suggesting a crucial promotive role of OVOS2 in the pathogenesis and progression of CMM. The involved mechanisms are at least partly associated with the overactivation of FAK/MAPK/ERK and FAK/PI3K/AKT signals.

Fig 1. OVOS2 expression in different melanoma cell lines.

(a) OVOS2 mRNA level in the four melanoma cell lines: A375, M14, MV3, and Sk-mel-1. The expression levels are shown relative to the primary cultured melanocytes. (b) OVOS2 protein expression analyzed using western blotting, with β-actin as the loading control. (c) OVOS2 protein expression measured by immunocytochemical analysis using polyclonal anti-OVOS2 antibody (magnification of A375 and MV3: 100×; M14 and MC: 200×). Sk-mel-1 was not tested for using immunocytochemistry because of being suspension-cultured.

Fig 2. OVOS2 expression in A375 cells transfected with or without OVOS2-shRNA.

(a) A decrease of OVOS2 mRNA was observed after transfection with four OVOS2-shRNAs (S1, S2, S3, and S4) in A375. The expression levels are shown relative to A375 cells transfected with the negative control-shRNA (NC). Then the stable transfectants of OVOS2-shRNA1 (hereafter referred to as OVOS2-shRNA) were chosen for further study. (b) OVOS2 protein expression measured by immunocytochemical analysis in untreated A375 cells (100×), A375 cells transfected with NC-shRNA (100×), and A375 cells transfected with OVOS2-shRNA (S1) (200×).

Fig 3. Effect of OVOS2 downregulation on cell proliferation, cell cycle, and apoptosis of A375.

There are representative results from three independent experiments. (a) The cell proliferation rate evaluated by MTT assay was significantly inhibited after 72 h of incubation in A375 cells transfected with the OVOS2-shRNAs, while no significant difference was observed at 24 or 48 h (**P < 0.01, ANOVA, LSD multiple comparison). (b) Results of cell number counting test. The proliferative rate at 72 h significantly decreased in A375/OVOS2–shRNA compared with that in A375/NC–shRNA and untreated A375 cell groups (**P < 0.01, rANOVA, LSD multiple comparison). (c) The colony formation assay showed the colony forming activity during the 14 d culture of A375 cells, A375/NC-shRNA and A375/OVOS2-shRNA (**P < 0.01, ANOVA, LSD multiple comparison). (d) Flow cytometric analysis showed significant accumulation of G2/M phase fraction at 72 h in A375 transfected with OVOS2-shRNA (**P < 0.01, *P < 0.05, ANOVA, LSD multiple comparison). (e) Flow cytometer analysis by staining with AnnexinV and 7-AAD. In the scatter plots, the left quadrant represents viable cells, and the right quadrant represents apoptotic cells. The results showed an unaffected apoptosis rate at 48 h and a non-significant increased apoptosis rate at 72 h in A375/OVOS2-shRNA, compared with A375 cell and A375/NC-shRNA (P > 0.05, ANOVA). (f) Results of Caspase-3 activity analysis. Downregulation of OVOS2 did not significantly affect the percentage of apoptotic cells after 48 (P>0.05, ANOVA) or 72 h culture (P = 0.05, ANOVA).

Fig 4. Effect of OVOS2 downregulation on the cell invasive behavior of A375.

(a) The enhanced cell-matrix adhesion rate of A375 cells transfected with OVOS2-shRNA was shown (*P < 0.05, P = 0.007 and P = 0.026, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). The average was calculated from three independent experiments and presented with standard deviation. (b) The wound healing assay showed the inhibition of cell motility in A375 cells following OVOS2 downregulation with shRNA (**P<0.01, repeated measure of ANOVA). Representative results from three independent experiments were shown (100×). (c) The inhibition of the transmigration ability during 24 h culture of A375 cells transfected with OVOS2-shRNA was shown using the Transwell migration study (*P < 0.05, P = 0.016 and P = 0.02, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison); and a nonsignificant decrease of invasion ability during 48 h culture of A375 cells transfected with OVOS2-shRNA was shown by the Matrigel invasion assay (P = 0.453, ANOVA). Representative results from three independent experiments (200 ×).

Fig 5. Western blot results.

(a) The levels of cyclin A, cyclin B, cyclin D1, and CDK2 were reduced in A375 cells transfected with OVOS2-shRNA; (b) The downregulated expression of N-cadherin accompanied with the upregulated expression of E-cadherin and β-catenin were observed in A375 cells transfected with OVOS2-shRNA; (c) The expression of p-FAK, p-AKT, and p-ERK were reduced in A375 cells transfected with OVOS2-shRNA; (d) The increased production of MMP-2 was observed in A375 transfected with OVOS2-shRNA; (e) GAPDH was used as the reference.

Fig 6. BALB/C nude mouse subcutaneous transplantation tumor experiment and immunohistochemistry of the transplanted tumors.

(a) All the mice from the three groups developed tumors. OVOS2 downregulation significantly decreased tumor growth and one tumor spontaneously regressed in the A375/OVOS2-shRNA group. (b) The tumor growth was significantly inhibited in the A375/OVOS2-shRNA group during the observation period (*P < 0.05, P = 0.011 and P = 0.03, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; repeated measure of ANOVA); the weight of tumors in the A375/OVOS2-shRNA group was also significantly reduced (*P < 0.05, P = 0.019 and P = 0.012, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). The results are shown as means ± SD. (c) H&E staining of the three groups (100×). The histopathologic characteristics of the xenograft tumors among the three groups were similar. (d) Number of abnormal mitoses of transplanted tumors in every high power field. The number of abnormal mitoses was much less in the A375/OVOS2–shRNA tumors compared with those in the A375 cell and A375/NC–shRNA tumors (*P < 0.05, P = 0.002 and P = 0.023, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). (e) The downregulation of OVOS2 in A375 cells dramatically decreased the percentage of Ki-67 positive cells as shown by immunohistochemistry study (**P < 0.01, ANOVA, LSD multiple comparison). (f) Immunohistochemistry staining of OVOS2 expression in transplanted tumors. OVOS2 was expressed in tumors induced by A375 and A375/NC–shRNA cells, but it was absent in one of the A375/OVOS2–shRNA cell-induced tumors, and the expression level in the group of A375/OVOS2–shRNA was significantly decreased (*P < 0.05, P = 0.009 and P = 0.033, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; Kruskal–Wallis test). (g) The expression of E-cadherin and β-catenin in the xenograft tumors showed that both E-cadherin and β-catenin were significantly upregulated in the A375/OVOS2-shRNA tumor (*P < 0.05, P = 0.015 and P = 0.02 for E-cadherin, P = 0.033 and P = 0.04 for β-catenin, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). (h) Immunohistochemistry staining for apoptosis indexes of Bax and Bcl-2in transplanted tumors. The ratio of Bax to Bcl-2 was higher in the A375/OVOS2–shRNA group than that in the A375 and A375/NC–shRNA groups; but not reached statistical significant difference (P>0.05, Kruskal–Wallis test).