Effects of silencing endothelin-1 on invasion and vascular formation in lung cancer

Abstract

Endothelin-1 (ET-1), which exists not only in the vascular endothelium but is also widely present in various tissues and cells, is an important cardiovascular regulatory factor that serves an important role in maintaining the basal vascular tone and homeostasis in the cardiovascular system. In the present study, the ET-1 gene was silenced by RNA interference, and the effects on lung cancer cell proliferation and tumor cell invasion were then detected by Cell Counting kit-8 and Transwell assays. In addition, the expression of apoptosis, growth and invasion-associated proteins, including RhoA/C, vascular endothelial growth factor, pigment epithelium-derived factor, AKT, E-cadherin and cyclooxygenase-2 was evaluated by western blotting upon silencing ET-1. In the present study, Endostar, a recombinant human endostatin injectable drug, was also used, and it was assessed whether the sensitivity of tumor cells to this drug could be increased by silencing ET-1. Both in vivo and in vivo tests were carried out in the present study. The experimental data indicated that ET-1 silencing can inhibit tumor cell proliferation and invasion, particularly in the presence of Endostar.

Keywords: A549; Endostar; endothelin-1; invasion; proliferation.

Figure 1.

shRNA ET-1 was successfully transfected into A549 Cells. (A and B) To evaluate the transfection efficiency, the reporter gene enhanced green fluorescent protein was observed under (A) white light and (B) green fluorescence (magnification, ×100). (C) The expression of shRNA ET-1 in A549 cells was determined by reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± SD, n=3. *P<0.05 compared with the vector group. (D) The proliferation of different groups of A549 cells was detected by Cell Counting kit-8 assay. Data are presented as the mean ± SD, n=3. *P<0.05 compared with the vector group. sh, short hairpin; ET, endothelin; mRNA, messenger RNA; SD, standard deviation.

Figure 2.

(A and B) Invasion of A549 cells subjected to different treatments. Data are presented as the mean ± standard deviation, n=3. *P<0.05 compared with the vector group; ^#P<0.05 compared with the vector + Endostar group. sh, short hairpin; ET, endothelin.

Figure 3.

Relative protein expression, as detected by western blotting after treatment for 48 h. Data are presented as the mean ± standard deviation, n=3. *P<0.05 compared with the vector group; ^#P<0.05 compared with the vector + Endostar group. VEGF, vascular endothelial growth factor; p, phosphorylated; Cox, cyclooxygenase; PEDF, pigment epithelium-derived factor; sh, short hairpin; ET, endothelin.

Figure 4.

Effect of ET-1 RNA interference in vivo. (A) Mice injected with empty vector. (B) Tumor volume was measured. (C) Mice injected with ET-1-silenced A549 cells. (D and E) Tumors were dissected, and their (D) volume and (E) weight were measured with a vernier caliper. ***P<0.001, *P<0.05 compared with A549 cells. sh, short hairpin; ET, endothelin.