Effects of osteopontin RNA interference on proliferation and apoptosis of rat vascular smooth muscle Cells

Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying osteopontin (OPN) gene of rat,and to investigate its effects on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs) by silencing OPN.Methods Four oligonucleotides targeting OPN gene were synthesized and cloned into lentivirus vector PLKO.1.The shRNA lentiviral vector with the best transfection efficiency was detected and identified,which was transfected into rat VSMCs.After RNA interference ( RNAi)-mediated knockdown of rat OPN,the mRNA and protein levels of OPN in VSMCs were assessed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.The effects of silencing OPN on the proliferation and apoptosis of VSMCs were examined by methyl thiazol tetrazolium (MTT) assay and flow cytometry.Results The recombinant lentivirus vector PLKO.1-OPN-shRNA was constructed successfully.The recombinant lentivirus PLKO.1-OPN-shRNA knocked down the expression of OPN mRNA and protein in VSMCs dramatically,especially PLKO.1-OPN2-shRNA,whose transfection efficiency was more than 90％.The proliferation rate of VSMCs transfected with PLKO.1-OPN2-shRNA (A value =0.365 ±0.011 ) was significantly lower than un-transfected group (A value =0.941 ±0.028) and negative control group (A value =0.941 ± 0.040,P ＜ 0.05 ).Meantime the early apoptosis rate (20.44 ± 2.69)％ and late apoptosis rate ( 16.79 ± 1.01 )％ were significantly higher in transfection group than un-transfected group and negative control group ( P ＜ 0.01 ).Conclusion The recombinant lentivirus vector expressing shRNA of targeting OPN is constructed successfully.And shRNA with the best transfection efficiency is PLKO.1-OPN2-shRNA,which can inhibit the proliferation of VSMCs,and promote the cell apoptosis. Key words: Osteopontin; Vascular smooth muscle cells; Vascular restenosis; RNA interference; Lentivirus