Nicotinamide Phosphoribosyl Transferase (Nampt) Is a Target of MicroRNA-26b in Colorectal Cancer Cells

<div><p>A number of cancers show increased expression of Nicotinamide phosphoribosyl transferase (Nampt). However, the mechanism through which Nampt is upregulated is unclear. In our study, we found that the Nampt-specific chemical inhibitor FK866 significantly inhibited cell survival and reduced nicotinamide adenine dinucleotide (NAD) levels in LoVo and SW480 cell lines. Bioinformatics analyses suggested that miR-26b targets Nampt mRNA. We identified Nampt as a new target of miR-26b and demonstrated that miR-26b inhibits Nampt expression at the protein and mRNA levels by binding to the Nampt 3′-UTR. Moreover, we found that miR-26b was down regulated in cancer tissues relative to that in adjacent normal tissues in 18 colorectal cancer patients. A statistically significant inverse correlation between miR-26b and Nampt expression was observed in samples from colorectal cancer patients and in 5 colorectal cell lines (HT-29, SW480, SW1116, LoVo, and HCT116). In addition, over expression of miR-26b strongly inhibited LoVo cell survival and invasion, an effect partially abrogated by the addition of NAD. In conclusion, this study demonstrated that the NAD-salvaging biosynthesis pathway involving Nampt might play a role in colorectal cancer cell survival. MiR-26b may serve as a tumor suppressor by targeting Nampt.</p></div

An example of evolutionary cycling.

<p>(a) Time series plots obtained through simulation of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">Model (9)</a> with initial condition (<i>x</i>₁, <i>x</i>₂) = (0.3, 0.8) and <i>a</i>₂ = 5.0. Predators and prey evolve to a stable limit cycle. (b) Equilibrium population density of prey species as the traits (<i>x</i>₁, <i>x</i>₂) evolve. (c) Equilibrium population density of predator species as the traits (<i>x</i>₁, <i>x</i>₂) evolve. The other parameter values are the same as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g002" target="_blank">2</a>.</p

Effects of miR-26b and NAD+ on the cell survival and invasiveness of colorectal cancer cells.

<p>A. LoVo cells were treated with miR-26b mimics et al., and cell viability was determined using Cell Counting Kit-8. B. The quantification of apoptotic cells induced by miR-26b mimics (with and without NAD) or miR-26b inhibitor was confirmed by flow cytometry analysis. The sub-G1 contents were designated apoptotic cells. C. Representative micrographs of cell invasion assays (left) and the quantification (right) from different treatments (*p<0.01). The stained invasive cells were photographed under an inverted light microscope (100× magnification). *: versus negative control group; **: compared with 2 treatment groups. Each vertical bar represents the mean ± SD of triplicate determinations. For comparison of 2 groups, a two-tailed, unpaired t-test was used.</p

The effects of miR-26b on Nampt expression.

<p>A. Quantitative real-time PCR of Nampt in SW480 cell lines after transfection with different concentrations of miR-26b mimics or inhibitors. B and C. Western blotting of Nampt in SW480 cell lines after transfection with different concentrations of miR-26b mimics or inhibitors. D. The relative levels of NAD in SW480 cell lines after transfection with different concentrations of miR-26b mimics or inhibitors. E. Luciferase reporter assays validating the direct interaction of miR-26b with the 3′-UTR of Nampt. Nampt-3′-UTR-pEZX (or Nampt-3′-UTR-mut-pEZX) was cotransfected with miR-26b mimic or negative control into SW480 cells. *: versus negative control group; **: compared with 2 treatment groups. Each vertical bar represents the mean ± SD of triplicate determinations. For comparison of 2 groups, a two-tailed, unpaired t-test was used.</p

Primer sequences (forward and reverse).

<p>Primer sequences (forward and reverse).</p

The concave-convex capture rate function and two trade-off functions.

<p>(a) Asymmetric capture rate <i>a</i>(<i>x</i>₁ − <i>x</i>₂) as a function of phenotypic difference (<i>x</i>₁ − <i>x</i>₂), where <i>a</i>₀ = 0.1, <i>a</i>₁ = 1.0. (b) The growth rate of prey <i>r</i>(<i>x</i>₁) (trade-off function), where <i>r</i>₀ = 0.5, <i>r</i>₁ = 1.5, <i>r</i>₂ = 1.5. (c) The death rate of predators <i>m</i>(<i>x</i>₂) (trade-off function), where <i>m</i>₀ = 0.2, <i>m</i>₁ = 0.5, <i>m</i>₂ = 0.9.</p

Evolutionary branching and evolutionary murder of predator species.

<p>(a) Traits coevolution plot when <i>a</i>₂ = 2.21. The vector fields obtained from deterministic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">Model (9)</a> indicate directions of coevolution of traits <i>x</i>₁ and <i>x</i>₂. The black curve and red curve indicate respectively isoclines of traits <i>x</i>₁ and <i>x</i>₂. The solid curves indicate evolutionarily singular strategies which are evolutionarily stable, while the dashed curve indicates evolutionarily singular strategy which is not evolutionarily stable. The grey region is feasible phenotype space, in which predator-prey coevolution can occur. (b) Pairwise invasibility plot (PIP) for fixed prey strategy . (c) Mutual invasibility plot (MIP) for fixed prey strategy . (d) Simulated evolutionary tree obtained through simulation of Models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">9</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e074" target="_blank">29</a>) with initial condition (<i>x</i>₁, <i>x</i>₂) = (0.3, 0.8) and <i>a</i>₂ = 2.21. (e) Equilibrium population density of prey species along the coevolutionary trajectory. (f) Equilibrium population densities of predator species along the coevolutionary trajectory. At evolutionary time <i>τ</i>₂, the equilibrium population density of predator 1 becomes zero. The red curve indicates total equilibrium population density of the two predator species. Parameter values: <i>k</i> = 0.01, <i>b</i> = 0.25, <i>c</i> = 0.001, <i>μ</i>₁ = <i>μ</i>₂ = <i>μ</i>₂₁ = <i>μ</i>₂₂ = 0.03, <i>σ</i>₁ = <i>σ</i>₂ = <i>σ</i>₂₁ = <i>σ</i>₂₂ = 0.03. The other parameter values are the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g001" target="_blank">Fig 1</a>.</p

Nampt mRNA levels were inversely correlated with miR-26b levels in colorectal cancer cell lines and patient samples.

<p>A. miR-26b, Nampt mRNA and NAD+ levels were assayed in colorectal cancer cell lines. The expression of miR-26b was inversely correlated with Nampt expression in colorectal cancer cell lines; the expression of NAD+ was positive correlated with Nampt expression in colorectal cancer cell lines (A right). B. miR-26b and Nampt mRNA levels were assayed in 18 surgical specimens of human colorectal cancer tissues and adjacent normal colorectal tissues. Significantly upregulated Nampt levels in colorectal cancer tissues are shown relative to Nampt levels in adjacent normal colorectal tissues (fold changes >6); significantly downregulated miR-26b levels in colorectal cancer tissues are shown relative to miR-26b levels in adjacent normal colorectal tissues (fold changes >2). C. The expression of miR-26b was inversely correlated with Nampt expression in colorectal cancer tissues (C left), and adjacent normal colorectal tissues (C right). Nampt mRNA levels were assayed by real-time RT-PCR and normalized to GAPDH. The miR-26b levels were assayed by real-time RT-PCR and normalized to U6. Each vertical bar represents the mean ± SD of triplicate determinations. For comparison of 2 groups, a two-tailed, unpaired t-test was used.</p

An example of continuously stable strategy.

<p>(a) Time series plots obtained through simulation of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">Model (9)</a> with initial condition (<i>x</i>₁, <i>x</i>₂) = (0.3, 0.8) and <i>a</i>₂ = 2.5. (b) Fitness landscape plots when and <i>a</i>₂ = 2.5. (c) Equilibrium population density of prey species as the traits (<i>x</i>₁, <i>x</i>₂) evolve. (d) Equilibrium population density of predator species as the traits (<i>x</i>₁, <i>x</i>₂) evolve. The other parameter values are the same as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g002" target="_blank">2</a>.</p

Evolutionary branching and evolutionarily stable coexistence of prey species.

<p>(a) Traits coevolution plot when <i>a</i>₂ = 3.8. The vector fields obtained from deterministic <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">Model (9)</a> indicate directions of coevolution of traits <i>x</i>₁ and <i>x</i>₂. The black curve and red curve indicate respectively isoclines of traits <i>x</i>₁ and <i>x</i>₂. The solid curves indicate evolutionarily singular strategies which are evolutionarily stable, while the dashed curves indicate evolutionarily singular strategies which are not evolutionarily stable. The grey region is feasible phenotype space, in which predator-prey coevolution can occur. (b) Pairwise invasibility plot (PIP) for fixed predator strategy . (c) Mutual invasibility plot (MIP) for fixed predator strategy . (d) Simulated evolutionary tree obtained through simulation of Models (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e010" target="_blank">9</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.e059" target="_blank">23</a>) with initial condition (<i>x</i>₁, <i>x</i>₂) = (0.3, 0.8) and <i>a</i>₂ = 3.8. (e) Fitness landscape plots when and <i>a</i>₂ = 3.8. (f) Equilibrium population densities of prey species along the coevolutionary trajectory. The red curve indicates total equilibrium population density of the two prey species. (g) Equilibrium population density of predator species along the coevolutionary trajectory. Parameter values: <i>μ</i>₁ = <i>μ</i>₂ = <i>μ</i>₁₁ = <i>μ</i>₁₂ = 0.03, <i>σ</i>₁ = <i>σ</i>₂ = <i>σ</i>₁₁ = <i>σ</i>₁₂ = 0.03. The other parameter values are the same as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163753#pone.0163753.g002" target="_blank">2</a>.</p