Effects of small interfering RNA silencing in epidermal growth factor receptor on proliferation and migration of vascular smooth muscle cells

Abstract

Objective To investigate the effect of silencing epidermal growth factor receptor (EGFR) by small interfering RNA (siRNA) interference on the proliferation and migration of vascular smooth muscle cells (VSMCs), and the related mechanisms. Methods VSMCs of rats were isolated and cultured. VSMCs were infected with EGFR siRNA lentiviral vectors. The cells were divided into siRNA-EGFR group, siRNA-negative control group and blank control group. The cell proliferation abilities were detected by 5’-bromo-2’-deoxyuridine (5-BrdU) method. The cell migration abilities in different transfection groups were examined by Transwell chamber and scratch assays. The expression levels of EGFR, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 genes in different transfection groups were detected using real-time quantitative polymerase chain reaction (Real-time PCR) technology. The expression levels of EGFR, p-ERK, p-JNK and p-p38 proteins in different transfection groups were detected by Western blotting. Results The number of migration cells in the siRNA-EGFR group was 48.5±6.1, which were significantly lower than the siRNA-negative control group and blank control group, which were 86.2±7.2 and 88.3±7.6; the difference was statistically significant (F=37.261, P=0.000). Compared with 0 h, after 24 h, the migration rate of VSMC in the siRNA-EGFR group was (35.7±3.8)%, which was significantly lower than that of siRNA-EGFR group and blank group, which were (74.2±5.7)% and (75.5±6.1)%; the differences were statistically significant (F=40.315, P=0.000). The relative expression levels of ERK, JNK and p38 mRNA in the siRNA-EGFR group (0.53±0.09, 0.47±0.11, 0.41±0.07) were lower than the siRNA-negative control group (0.82±0.12, 0.64±0.10, 0.68±0.09) and blank control group (0.85±0.14, 0.66±0.13, 0.70±0.12), the differences were statistically significant (F=15.391, 12.817, 11.264, P=0.000, 0.000, 0.000). The relative expression levels of p-ERK, p-JNK and p-p38 proteins in the siRNA-EGFR group (0.46±0.09, 0.38±0.06, 0.33±0.06) were lower than the siRNA-negative control group (0.71±0.12, 0.57±0.08, 0.52±0.07) and blank control group (0.73±0.13, 0.56±0.07, 0.54±0.09), the differences were statistically significant (F=20.152, 14.375, 13.843, P=0.000, 0.000, 0.000). Conclusion Specific inhibition of EGFR could effectively reduce proliferation and migration capacities of VSMCs, which might be related to inhibition of mitogen-activated protein kinase signaling pathway. Key words: Vascular smooth muscle cells; Epidermal growth factor receptor; Small interference; Cell proliferation; Cell migration