Expression of Slit3/Robo signal pathway in mouse aortic smooth muscle cell and its impact on proliferation and migration

Abstract

To observe the expression of neural axon guidance molecules Slit3 and Robo receptors in mouse aortic smooth muscle cell(MASMC) and investigate the effect of exogenous Slit3 protein on migration and proliferation of MASMC. The primary cultured MASMC were identified by immunofluorescent assay. The expression of Slit3/Robo signal pathway was detected by RT-PCR and immunocytochemical staining. MASMC were divided into 6 groups: the negative control group (DMEM medium containing bovine serum albumin 86 μg/L), Slit3 0 μg/L group (DMEM medium without Slit3), Slit3 24 μg/L group (DMEM medium containing Slit3 24 μg/L), Slit3 40 μg/L group (DMEM medium containing Slit3 40 μg/L), Slit3 80 μg/L group (DMEM medium containing Slit3 80 μg/L) and the positive control group (DMEM medium containing platelet derived growth factor 10 μg/L). The effects of exogenous Slit3 on MASMC proliferation and migration were detected by CCK-8 and scratched cells and transwell chambers respectively. (1) The mRNA and protein expressions of Slit2, Slit3, Robo1 and Robo4 were detected in MASMC. mRNA level of Slit2 was lower than Slit3 (P<0.05) and there were no significant difference between mRNA level of Robo1 and Robo4. (2) The mitogenic responses of MASMC were significantly enhanced in Slit3 24 μg/L group, Slit3 40 μg/L group and Slit3 80 μg/L group compared with negative control group (1.13±0.04, 1.19±0.02, 1.18±0.08 and 0.64±0.10 respectively, all P<0.05). The mitogenic activity of MASMC was the strongest in Slit3 40 μg/L group (compared with positive control group 1.27±0.05, P>0.05). (3)The autonomous migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (cell scratch width were (0.40±0.03)cm, (0.32±0.03)cm, (0.30±0.02)cm and (0.49±0.01)cm respectively, all P<0.05). The autonomous migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group (0.22±0.01)cm, P>0.05). The transmembrane migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (the number of cell migration were 46.67±2.23, 65.33±3.43, 81.67±4.22 and 39.33±2.03 respectively, all P<0.05). The transmembrane migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group 84.00±2.02, P>0.05). Slit2, Slit3, Robo1 and Robo4 were expressed in MASMC, and exogenous Slit3 could promote proliferation and migration of MASMC in vitro.