The mechanism of phagocytosis regulation mediated by miR-1.
(A) miR-1 targets analysis. Clathrin heavy chain 1 gene (CLTC1) was a predicted target gene of miR-1. (B) The interaction between miR-1 and CLTC1 gene. RAW264.7 cells were simultaneously transfected with miR-1 or control (plasmid only) and the CLTC1 gene 3′ UTR, followed by a dual luciferase reporter assay. The CLTC1 gene 3′ UTR mutant was included in the assays. (C) Downregulation of endogenous CLTC1 gene by miR-1 in RAW264.7 cells. The miR-1 precursor and the negative control were transfected into RAW264.7 cells, respectively. Subsequently the CLTC1 mRNA was detected using real-time quantitative PCR (left) and the CLTC1 protein was examined with Western blot (right). In real-time PCR, the expression level of CLTC1 gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase gene. In Western blot, the antibodies used were indicated on the right. (D) The role of CLTC1 in phagocytosis of macrophages. RAW264.7. Cells were transfected with CLTC1-siRNA to silence the expression of CLTC1. CLTC1-siRNA-scrambled was used as a control. At 48 h after transfection, the expression of CLTC1 was detected with quantitative real-time PCR (left). At the same time, the phagocytosis percentages of RAW264.7 cells were evaluated using flow cytometry (right). (E) Model for miR-1-mediated pathway in phagocytosis. In all panels, the data referred to the means ± standard deviation of triplicate assays. Statistically significant differences between treatments were indicated with asterisk (*, p<0.05).