Effect of sirolimus on cell proliferation and expression of origin recognition complex 1 in vascular smooth muscle cells of rats

Abstract

Objective To study the effect and mechanism of sirolimus on cell proliferation and expression of origin recognition complex 1(ORC1) in rat vascular smooth muscle cells(VSMCs) in vitro.Methods VSMCs were randomly divided into two groups,three bottles/well per group;synchronization of cells was achieved by serum starvation.Control group was cultured in DMEM with 10% FBS,and experimental group was cultured in DMEM with 10 μmol/L sirolimus plus 10% FBS.Expression of proliferating cell nuclear antigen(PCNA) protein was analyzed by immunocytochemistry,VSMC ultramicrostructure was observed under electron microscope,and expressions of P53,cyclin D1,cyclin A,and ORC1 mRNA and protein were displayed by RT-PCR and Western blotting analysis during 0-48 h of culture.Results Compared with control group,the positive rate of PCNA protein in experimental group was significantly decreased after 48 h culture([20±2.1]% vs [80±3.0]%,P0.05),P53 protein expression was significantly increased(P0.05),with evidence of pyknosis.The expression of cyclin D1 was slightly increased in the experimental group(P0.05),and the expression of cyclin A was significantly decreased after 48 h culture(P0.05).ORC1 mRNA expression in the control group was increased during 0-12 h and decreased during 24-48 h,with the expression peaked at 12 h.In the experimental group ORC1 mRNA expression was kept at a high level,with the expression at 48 h being significantly higher than that in the control group(P0.05);similar results were also found for ORC1 protein expression.Conclusion Sirolimus can inhibit the proliferation of VSMCs and promote their apoptosis.The inhibition of VSMCs proliferation may be through preventing cell transformation of G0/G1 phase to S phase and keeping cells at resting phase.The effect of ORC1 in cell cycle occurs at the upstream of sirolimus,which further supports that ORC1 participates in the initiation process of DNA replication.