Effects of silencing Eph A7 gene on angiogenesis of SMMC-7721 cell xenograft in nude mice

Abstract

Objective To investigate the effects of RNA interference targeting Eph A7 gene on angiogenesis of SMMC-7721 cell xenograft in nude mice. Methods Recombinant plasmid of Eph A7 gene-targeting small interfering RNA(siRNA) was transfected into SMMC-7721 cells by Lipofectamine TM 2000.The nude mice tumor model was established by subcutaneous injection of hepatic cancer cells in the left upper limb of the mice. The nude mice were then divided into transfected group, empty vector transfected group and untransfected group according to the different cells injected.The xenograft tumor tissues were obtained 35 days later.Real-time PCR assay was used to detect the expression of Eph A7 mRNA in tumor tissues in different groups.Immunohistochemistry and Western blot assay were used to detect the expression of vascular endothelial growth factor(VEGF) and Angiopoietin-2(Ang-2) protein in tumor tissues of different groups and the microvessel density(MVD)was calculated also. Results Real-time PCR results showed that compared with empty vector transfected group(0. 390 0±0. 086 3)and untransfected group(0. 421 9±0. 059 5), the expression of Eph A7 m RNA in transfected group(0. 191 1±0. 020 4)was remarkably suppressed.Immunohistochemistry showed that VEGF and Ang-2 protein positive staining cells in tumor tissues of transfected group were less and lighter staining.Western blot assay showed that compared with empty vector transfected group(0. 490 8±0. 084 4, 0. 510 8±0. 104 8) and untransfected group (0. 555 9±0. 075 4, 0. 564 0±0. 178 7), the expression VEGF and Ang-2 protein in transfected group(0. 360 9±0. 075 2,0. 334 7±0. 088 2) were remarkably suppressed(P<0. 05).Microvessel density calculation shows that the average value of MVD of the transfected group(42. 88±12. 80) was significantly lower than that of empty vector transfected group(60. 63±12. 13)and untransfected group(64. 38±10. 51)(P< 0. 05). Conclusion The inhibition of Eph A7 gene can down regulate the expression of VEGF and Ang-2 in SMMC-7721 hepatoma cell line.Eph A7 may regulate angiogenesis of hepatocellular carcinoma through a regulation of the expression of VEGF and Ang-2. Key words: Hepatocellular carcinoma; Eph A7; Vascular endothelial growth factor; Angiopoietin-2; Angiogenesis