Objective      To construct RNA interference (RNAi) lentiviral vector targeting murine transcription factor E2-2 gene and investigate its effect on the growth and proliferation of endothelial progenitor cells (EPCs).       Methods      Murine EPCs were isolated from bone marrow

cultured and identified. Three potential RNAi sites were screened based on the mRNA sequence of E2-2 gene and synthesized. The 3 synthesized siRNAs were transferred into the EPCs

and reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the inhibitory effect of the target gene so as to identify the optimal siRNA sequence. Then the short-hairpin RNA (shRNA) oligonucleotide targeting the optimal siRNA sequence was designed

synthesized and inserted into FT203-PLVX plasmid to construct FT203-PLVX/E2-2shRNA lentiviral vector. After identifying by sequencing

FT203-PLVX/E2-2shRNA plasmid was transferred into 293 cells to produce lentiviral particles. The EPCs were transfected with the lentiviral particles

and the expression of green fluorescent protein (GFP) in the transfected cells was observed with an inverted microscope. Cell count kit-8 (CCK-8) was used to test the growth and proliferation of the transfected cells. The expression of E2-2

proliferating cell nuclear antigen (PCNA) gene and its encoding protein in the transfected cells were quantitatively detected by RT-PCR and Western blotting.       Results      Murine EPCs were isolated from the bone marrow

cultured and identified. The optimal siRNA sequence CGTCAGCTAGTGTTTCTAA was confirmed. Sequencing results showed that lentiviral vector FT203-PLVX/E2-2shRNA was constructed successfully. The transfected cells significantly expressed GFP. The CCK-8 results revealed that the growth and proliferation of EPCs were faster after E2-2 gene silencing

especially from 48 h after transfection (P0.01). The results of RT-PCR

Western blotting and quantitative analysis showed that the lentiviral vector FT203-PLVX/E2-2shRNA effectively silenced E2-2 gene and up-regulated the expression of PCNA in the EPCs.       Conclusion      Murine EPCs are isolated from bone marrow

cultured and identified. The lentiviral vector targeting E2-2 gene can effectively silence E2-2 gene

up-regulate PCNA expression

and promote the growth and proliferation of the EPCs.