Knockdown of Toll-like receptor 2/4 by small interfering RNA suppresses the migration of osteoblasts

Objective To explore the effects of Toll-like receptor 2/4 (TLR2/TLR4) on the migration and proliferation of osteoblasts. Methods The expression of TLR2/TLR4 in osteoblasts was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Then the expression of TLR2/TLR4 in osteoblasts was suppressed using RNA interference technique. The influences on the migration and proliferation were analyzed by Transwell chamber assay and methyl thiazol tetrazolium (MTT) assay. Also, the effects of TLR2/TLR4 on the expression of nuclear factor-κB (NF-κB) p65 were detected. Results As shown by RT-PCR and Western blotting, the expression of TLR2/TLR4 significantly decreased in the TLR2/TLR4 small interfering RNA (siRNA)-transfected group. The inhibition rate of mRNA reached 94% and 84% respectively. The protein level of TLR2 and TLR4 was reduced to 48% and 65% respectively. The number of migrating cells in the TLR2-siRNA and TLR4-siRNA transfected groups was decreased to 76% and 59% compared to the control group. But the proliferation of osteoblasts was similar between those groups. Meanwhile, the siRNA-transfected group had lower expression level of NF-κB p65 in nucleus than the control group. The amount of NF-κB p65 in the nucleus was reduced to 47% and 75% in the TLR2-siRNA and TLR4-siRNA transfected groups compared to the control group. Conclusion siRNA inhibits the expression of TLR2/TLR4 effectively, weakens the activation of the NF-κB, and then inhibits the migration of the osteoblast. This study implicated that TLR2/TLR4 and NF-κB pathway may be involved in the migration of osteoblasts. Key words: Osteoblast; Toll-like receptor 2; Toll-like receptor 4; Proliferation; Small interfering RNA; Nuclear factor-κB