Role of Human Macrophage Migration Inhibitory Factor in Promoting Genes Expression Involved in Angiogenesis
SHAN Zhi-xin, YU Xi-yong, LIN Qiu-xiong, DENG Chun-yu, LIU Yuan, CAI Shi-xia, TAN Hong-hong, FU Yong-heng, LIN Shu-guang (Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou 510080, China)
[Objective] To investigate the potential role of macrophage migration inhibitory factor (MIF) in promoting angiogenic gene expression in human umbilical vascular endothelial cells (HVECs). [Methods] Using subcloning technique, the prokaryotic MIF expression plasmid pET22b-MIF was constructed. The MIF gene was induced by IPTG to express 6His-tag fusion protein in E. coli BL21 (DE3) and purified by HisTrap affinity columns. The collected recombinant MIF (rMIF) was renatured and the bioactivity of rMIF was tested by macrophage migration inhibition assay (MMI). The HVECs were incubated with 0, 30, 60, and 120 ng/mL rMIF for 12 h, and VEGF_(165), FGFR_3, MMP9, TGF-α and PDGF-α mRNA expression were determined by real-time quantitative PCR test. Using in vitro angiogenesis assay, the tube formation of HVECs induced by MIF was tested. [Results] pET22b-MIF was constructed correctly and transformed into E. coli BL21 (DE3). The rMIF was successfully expressed in E. coli in inclusive bodies. The result of MMI assay showed that the renatured rMIF inhibited the macrophage cells migration at the rate of 30％ (P＜0.05). The results of real-time quantitative PCR revealed that MIF specifically promoted VEGF_(165), FGFR_3, MMP9, TGF-α and PDGF-α genes expression in HVECs. The result of in vitro angiogenesis assay showed that MIF specifically induced tube formation of HVECs. [Conclusion] rMIF is successfully expressed in E. coli and rMIF can promote angiogenic factors expression in HUVECs.