The mechanism of phagocytosis regulation mediated by miR-1.

Abstract

<p>(A) miR-1 targets analysis. Clathrin heavy chain 1 gene (<i>CLTC1</i>) was a predicted target gene of miR-1. (B) The interaction between miR-1 and <i>CLTC1</i> gene. RAW264.7 cells were simultaneously transfected with miR-1 or control (plasmid only) and the <i>CLTC1</i> gene 3′ UTR, followed by a dual luciferase reporter assay. The <i>CLTC1</i> gene 3′ UTR mutant was included in the assays. (C) Downregulation of endogenous <i>CLTC1</i> 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 <i>CLTC1</i> mRNA was detected using real-time quantitative PCR (left) and the <i>CLTC1</i> protein was examined with Western blot (right). In real-time PCR, the expression level of <i>CLTC1</i> 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 <i>CLTC1</i> in phagocytosis of macrophages. RAW264.7. Cells were transfected with <i>CLTC1</i>-siRNA to silence the expression of <i>CLTC1</i>. <i>CLTC1</i>-siRNA-scrambled was used as a control. At 48 h after transfection, the expression of <i>CLTC1</i> 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 (*, <i>p</i><0.05).</p