The mechanism of phagocytosis regulation mediated by miR-1.

<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

The role of miR-1 in the regulation of phagocytosis in mammalian macrophages.

<p>(A) Expression levels of miR-1 and phagocytic activities in the isolated murine macrophage, the immortalized macrophage ANA-1 and the cancerous macrophage RAW264.7. The expression of miR-1 was quantified with real-time PCR (left). The phagocytic activity was evaluated using FITC-labeled <i>E. coli</i> (right). The data referred to the means ± standard deviations of triplicate assays. (B) Overexpression of miR-1 in RAW264.7 and ANA-1 cells. Cells were transfected with miR-1 precursor or control miRNA. At 24 h after transfection, the total RNAs were isolated from transfected cells and subjected to Northern blot. The expression level of miR-1 was normalized with U6. (C) Evaluation of phagocytic activities in RAW264.7 and ANA-1 cells against FITC-labeled <i>E. coli</i> by flow cytometry. Cells transfected with miR-1 precursor or control miRNA were challenged with FITC-labeled <i>E. coli</i>, followed by phagocytosis assays. (D) Knockdown of miR-1 in RAW264.7 and ANA-1 cells. The cells were transfected with AMO-miR-1 or AMO-miR-1-scrambled. Twenty four hours later, the total RNAs were isolated from transfected cells. The expression of miR-1 was determined by quantitative real-time PCR. (E) Phagocytosis percentages of RAW264.7 and ANA-1 cells treated with AMO-miR-1 or AMO-miR-1 -scrambled. At 24 h after transfection of AMOs, the phagocytosis was evaluated using FITC-labeled <i>E. coli</i>. In all panels, plotted data points referred to the means ± standard deviations of triplicate assays and asterisks represented statistically significant differences (**, <i>p</i><0.01; *, <i>p</i><0.05).</p

The regulation of phagocytosis mediated by miR-1 in shrimp.

<p>(A) Nucleotide sequences and modifications of the anti-miRNA-1 oligonucleotide (AMO-miR-1) and AMO-miR-1-scrambled. (B) Silencing of miR-1 expression in shrimp hemocytes <i>in vivo</i>. The miR-1-specific AMO (AMO-miR-1) and the negative control AMO-miR-1-scrambled were injected into shrimp, respectively. The shrimp hemocytes were subjected to Northern blot using miR-1 or U6 probe. (C) The effect of miR-1 expression silencing on phagocytic activity of shrimp hemocytes. The phagocytic activity of FITC-labeled WSSV was evaluated with flow cytometry. The plotted data points referred to the means ± standard deviations of triplicate assays and the asterisk represented statistically significant differences (*, <i>p</i><0.05). (D) The interaction between miR-1 and <i>CLTC1</i> gene. The miR-1 precursor and the plasmid EGFP-<i>CLTC1</i> or EGFP-<i>CLTC1</i>-mutant or EGFP were cotransfected into insect High Five cells. Then the fluorescence intensity of cells was monitored with a fluorescence microscope. The columns represented the means ± standard deviations of triplicate assays. The significant differences between treatments were indicated with asterisks (**, <i>p</i><0.01). (E) The effect of <i>CLTC1</i> on phagocytosis of shrimp hemocytes. The shrimp were injected with <i>CLTC1</i>-siRNA or <i>CLTC1</i>-siRNA- scrambled as a control. Then the shrimp were subjected to Northern blots (left) and phagocytosis assays (right). In Northern blots, the shrimp β-actin was used as a control. The significant differences between treatments were indicated with asterisk (*, <i>p</i><0.05). (F) Sequence alignment of miR-1 from six typical species. * indicated the identical nucleotides.</p