Objective: To investigate the effect of down-regulation of Kruppel like factor 4 (KLF4) by RNA interference on proliferation, apoptosis and phagocytosis in murine RAW264.7 macrophage cell line.
Methods: Stable KLF4 silencing in RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine KLF4 gene via liposome mediated transfection, followed by G418 selection. The efficacy of KLF4 silencing in G418 resistant cells was verified by fluorescent microcopy and Western blotting, respectively. Proliferative activity was analyzed by CCK-8 assay. Apoptosis and phagocytosis were evaluated by annexinV-FITC/PI staining and flow cytometry with assistant use of fluorescein-labeled E.coli K-12 particles, respectively.
Results: KLF4 protein expression was significantly down-regulated by the recombinant shRNA plasmid as compared with negative control (NC) plasmid transfection, inhibition rate being over 76%. From the third day, KLF4-silencing cells exhibited lower proliferative activity as compared with NC RAW264.7 cells (P<0.05). In resting state, apoptosis rate in wide type, NC and KLF4-silencing cells were 1.73%, 6.85% and 12.76%, respectively, and KLF4-silencing resulted in a statistical difference in apoptosis as compared with NC cells (P<0.05). Finally, phagocytic capability in wide type, NC and KLF4-silencing cells revealed by the mean fluorescence intensity of engulfed FITC-labeled E.coli particles were 122.0 ± 2.80, 48.97 ± 5.69 and 80.10 ± 4.61, respectively, and compared with NC cells, KLF4-silencing cells had a significant increase in phagocytosis (P<0.05).
Conclusion: KLF4 gene silencing inhibits RAW264.7 macrophage proliferation, increase apoptosis and enhance phagocytic function in resting state, which provides a novel tool for revealing the role of KLF4 in macrophage immunity.