Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

Abstract

Background: Vascular endothelial growth factor (VEGF)-A and VEGF-C are two important molecules involving in tumor development and metastasis via angiogenesis and lymphangiogenesis. However, the combined effect of VEGF-A and VEGF-C on the growth of gastric cancer (GC) is not clear.

Methods: The correlations of VEGF-A and VEGF-C expressions with clinicopathologic parameters and prognosis were evaluated in patients with GC. Furthermore, lentivirus-mediated RNA interfering (RNAi) targeting VEGF-A and/or VEGF-C was employed to silence their expressions in SGC7901 GC cell line. Cell proliferation and apoptosis were measured in vitro. Suppressive effect lentivirus-mediated VEGF-A and/or VEGF-C silencing on GC growth was evaluated in GC bearing mice.

Results: The patients with high expression of both VEGF-A and VEGF-C (A+C+) had larger tumor size, higher peritumoral lymphatic vessel density(P-LVD), microvessel density(MVD), lymphatic vessel invasion (LVI), lymph node(LN) metastasis, and worse prognosis than those with low expression of both VEGF-A and VEGF-C (P<0.05). Lentivirus-mediated RNAi significantly reduced the mRNA and protein expression of VEGF-A and VEGF-C in the SGC7901 cells. The Lenti-miRNA-VEGF-A+VEGF-C significantly inhibited the cell proliferation and tumor growth, compared with Lenti-miRNA-VEGF-A or Lenti-miRNA-VEGF-C (P<0.05). In addition, Lenti-miRNA- VEGF-A+VEGF-C markedly lowered the tumor size in vivo in comparison with Lenti-miRNA-VEGF-A or Lenti-miRNA-VEGF-C (P<0.05).

Conclusion: Expressions of both VEGF-A and VEGF-C predict worse prognosis of GC patients. Combined silencing of VEGF-A and VEGF-C markedly suppresses cancer growth than silencing of VEGF-A or VEGF-C. Thus, to inhibit the expressions of VEGF-A and VEGF-C may become a novel strategy for the treatment of GC.

Keywords: Vascular endothelial growth factor-A; gastric cancer; prognosis; tumor growth; vascular endothelial growth factor-C.

Figure 1

Detection of expressions of VEGF-A and VEGF-C in GC by immunohistochemistry and double immunohistochemical staining for D2-40 and CD34. A. VEGF-A expression in the cytoplasm (×200). B. VEGF-C expression in the cytoplasm (×400). C. The lymphatic vessels were clearly distinguished from blood vessels after double immunohistochemical staining for D2-40 (dark purple) and CD34 (intense red) (×400).

Figure 2

Relationship between expressions of VEGF-A and VEGF-C and overall survival.

Figure 3

Expression of VEGF-A or VEGF-C in SGC7901 GC cells transduced with pCMV-VEGF-A miRNA or pCMV-VEGF-C miRNA expression plasmids at 48 h (*P<.05, **P<.01). mRNA and protein expressions of VEGF-A in SGC7901 cells transduced with pCMV-VEGF-A miRNA expression plasmids. A. Real-time qPCR showed VEGF-A mRNA expression in pCMV-VEGF-A miRNA-4 transduced cells was significantly reduced by 85.6% when compared with parental SGC7901 cells. C. Western blot assay. E. VEGF-A protein expression in pCMV-VEGF-A miRNA-4 transduced cells was significantly reduced by 89.8% when compared with parental SGC7901 cells. mRNA and protein expressions of VEGF-C in SGC7901 cells transduced with pCMV-VEGF-C miRNA expression plasmids. B. Real-time qPCR assay showed VEGF-C mRNA expression in pCMV-VEGF-C miRNA-1 transduced cells was significantly reduced by 89.8% when compared with parental SGC7901 cells. D. Western blot assay. F. VEGF-C protein expression in pCMV-VEGF-C miRNA-1 transduced cells was significantly reduced by 60.4% when compared with parental SGC7901 cells.

Figure 4

Combined silencing of both VEGF-A and VEGF-C significantly suppressed the cell proliferation and induced the apoptosis of GA cells. A. Cell proliferation was significantly inhibited in SGC7901 cells after infection with Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C vectors as measured by MTT assay, compared with control groups (*P<.05, **P<.01). B. Cells infected with Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C vectors had the highest apoptosis rate detected by apoptosis assay, compared with control groups (*P<.05, **P<.01).

Figure 5

Combined silencing of VEGF-A and VEGF-C significantly inhibited cancer growth in vivo. A. SGC7901; B. Lenti-miRNA-neg; C. Lenti-miRNA-VEGF-A; D. Lenti-miRNA- VEGF-C; E. Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C. F. Silencing of VEGF-A or VEGF-C in SGC7901 cells could significantly inhibit the cancer growth, when compared with the parental cells or cells infected with Lenti-miRNA-neg (*P<.05, **P<.01). The combined silencing of VEGF-A and VEGF-C could more effectively suppress the GC growth than the silencing of VEGF-A or VEGF-C alone (*P<.05, **P<.01).

Figure 6

Effects of Lenti-miRNA-VEGF-A and/or Lenti-miRNA-VEGF-C on cancer growth in GC bearing nude mice. A. Cancer volumes were significantly reduced in mice treated with Lenti-miRNA-VEGF-A, Lenti-miRNA-VEGF-C, or Lenti-miRNA-VEGF-A +VEGF-C, as compared to those treated with Lenti-miRNA–neg or PBS (*P<.05, **P<.01). B. Cancers in the mice treated with Lenti-miRNA-VEGF-A + VEGF-C had the smallest volumes than those treated with Lenti-miRNA-VEGF-A or Lenti-miRNA–VEGF-C (*P<.05, **P<.01).