Abstract:

Objective To observe the effect of short hairpin RNA （shRNA）-mediated vascular endothelial growth factor （VEGF） gene silencing on the growth of human skin squamous cell carcinoma（SCC） xenografts in nude mice. Methods Two eukaryotic expression plasmids targeting VEGF gene, including psilencer-VEGF1-shRNA （VEGF-s1） and psilencer-VEGF2-shRNA （VEGF-s2）, as well as one negative control plasmid containing random target sequence （psilencer-Target-off-shRNA, T-off）, were chemically synthesized, and transfected into a human skin SCC cell line A431 to develop stably transfected cell lines. Real time quantitative PCR （RT-qPCR） and double-antibody sandwich enzyme-linked immunosorbent assay （ELISA） were carried out to measure the expression of VEGF mRNA and protein respectively in A431 cells. Twelve nude mice were divided into 4 goups to be subcutaneously inoculated in the axillary region with untransfected A431 cells as well as A431 cells transfected with VEGF-s1, VEGF-s2 and T-off, respectively. The tumor growth was observed in nude mice every 5 days. Twenty days after the inoculation, the mice were sacrificed, and transplanted tumors were obtained from the mice and subjected to an immunohistochemical study for the measurement of VEGF，proliferating cell nuclear antigen （PCNA） and CD34 expression. Data were statistically analyzed by using the Stata 7.0 software, and t test was conducted to compare the differences between groups. Results The mRNA and protein expression levels of VEGF were significantly lower in A431 cells transfected with VEGF-s1 and VEGF-s2 than in untransfected A431 cells （27.85 ± 3.95 and 24.69 ± 2.83 vs. 54.06 ± 6.38, t = 6.05, 7.29, both P ＜ 0.01; 32.67 ± 2.52 and 29.27 ± 1.10 vs. 52.85 ± 2.23, t = 8.04 and 11.53, both P ＜ 0.01）. Twenty days after the inoculation, the volume and weight of xenografted tumors in mice inoculated with VEGF-s1- and VEGF-s2-transfected A431 cells were significantly lower than those in mice with untransfected A431 cells （（192.50 ± 10.90） mm3 and （203.67 ± 3.21） mm3 vs. （272.00 ± 21.07） mm3, t = 5.80 and 5.55, both P ＜ 0.01; （0.05 ± 0.03） g and （0.13 ± 0.04） g vs. （0.25 ± 0.02） g, t = 9.60 and 4.64, both P ＜ 0.01）. Decreased expression rate of VEGF, PCNA and number of CD34-positive vessels were observed in the xenografted tumor tissue from mice inoculated with VEGF-s1- and VEGF-s2-transfected A431 cells compared with that from mice with untransfected A431 cells （52.00% ± 2.00% and 56.67% ± 3.06% vs. 70.00% ± 2.00%，both P < 0.01; 37.01% ± 2.41% and 33.94% ± 3.25% vs. 72.11% ± 3.02%, both P < 0.01; 2.05 ± 0.07 and 1.72 ± 0.10 vs. 4.01 ± 1.27, both P < 0.01）. No significant differences were observed in the above parameters between cells transfected with VEGF-s1- and VEGF-s2-transfected A431 cells, between untransfected and T-off-transfected A431 cells, between tumor xenografts derived from VEGF-s1- and VEGF-s2-transfected A431 cells, or between tumor xenografts derived from untransfected and T-off-transfected A431 cells （all P > 0.05）. Conclusions The shRNA targeting VEGF gene can significantly inhibit the expression of VEGF in A431 cells and A431-derived tumor xenografts in nude mice, in turn suppress the growth and attenuate the malignant phenotype of tumor.

Key words: VEGF