Effects of Annexin-A1 gene silencing induced by siRNA on the growth and migration of BV-2 cells and its possible mechanisms

Abstract

Objective To investigate the effects of Annexin-A1 (Anxa1) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms. Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells. The efficiency of siRNA-induced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay. MTT assay was performed to measure the proliferation of BV-2 cells with silenced expression of Anxa1 gene. Flow cytometry with Annexin V-FITC/PI double staining was used to detect the apoptosis rate of BV-2 cells. Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells. Western blot assay was performed to detect the expression of signaling proteins related to cell cycle and migration. Results Compared with the siRNA negative control (siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9±2.1)%, (23.1±3.6)% and (42.4±1.7)% vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05]. The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)% and control group (4.3±0.2)%. Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as compared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01). Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells. Conclusion The growth and migration of BV-2 cells were significantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways. Key words: RNA interference; Annexin-A1; BV-2 cell; Cell growth; Cell migration