Title: Effect of silencing of toll like receptor-4 gene by RNA interference on vascular smooth muscle cells proliferation
Abstract: Objective
To explore the effect and possible mechanisms on vascular smooth muscle cells (VSMC) proliferation of silencing of toll like receptor-4 (TLR4) gene using small interfering RNA (siRNA).
Methods
The recombinants of pSilence2.1-siTLR4 and the negative control plasmid pSilence2.1-NC were transfected to VSMC. VSMC expressing plasmid stably was collected and TLR4 mRNA and protein expression were detected. The VSMC was treated with lipopolysaccharide (LPS) as specific ligand of TLR4, the mRNA and protein expression lever of TLR4, Nuclear factor κB (NF-κB) was examined. Cell proliferation and cell cycle distribution were evaluated by methyl thiazol tetrazolium (MTT) colorimetry and flow cytometry.The statistical significance of differences between groups was calculated using one-way analysis of variance (ANOVA).
Results
The TLR4 mRNA and protein expression of siTLR4 group was significantly down-regulated to compare with the control and negative control plasmid (NC) group (FmRNA=111.300, FPro=274.500, P<0.01). VSMC growth in siTLR4 group was significantly slower compared with the control and the NC group when incubated with LPS, there were statistically significant in 72 h and 120 h (F72 h=5.920, F120 h=9.747, P<0.05). The proportion increased in G0/G1 phase in siTLR4 group compared with the control and NC group stimulating by LPS. The mRNA and protein expression of TLR4 and NF-κB in all groups were increased when incubated with LPS, which of the siTLR4 group was still significantly lower than the normal group and NC group (mRNA FTLR4=79.900, FNF-κB=211.900, Protein FTLR4=33.350, F NF-κB=136.900, P<0.05).
Conclusion
SiRNA-TLR4 reduced TLR4 mRNA and protein expression in VSMC and down-regulated LPS-induced NF-κB activation. siRNA-TLR4 can inhibit proliferation and growth of VSMC by arresting cell cycle in G0/G1 phase.
Key words:
Toll like receptor-4; RNA interference; Vascular smooth muscle cells; Proliferation; Cell cycle
Publication Year: 2019
Publication Date: 2019-06-08
Language: en
Type: article
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