Research ArticleCancer

Regulation of autophagy, NF-κB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells

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Sci. Signal.  12 Sep 2017:
Vol. 10, Issue 496, eaam6291
DOI: 10.1126/scisignal.aam6291
  • Fig. 1 KRAS oncogene dependency and epithelial-mesenchymal phenotypes are associated with a miRNA expression signature in NSCLC cell lines.

    (A) Volcano plot of differential miRNA expression based on quantitative PCR (qPCR)–based profiling in six representative KRAS mutant NSCLC cell lines. Fold expression differences were calculated by comparing average expression levels in three KRAS-dependent/epithelial (KE-correlated) cell lines to three KRAS-independent/mesenchymal (KM-correlated) cell lines. Student’s t test–derived P values are plotted on a −log scale on the horizontal axis (two-sided test with unequal variances). (B) Heat map representation of differential miRNA expression levels (P < 0.1) in KE versus KM subtype cell lines, as used in the volcano plot in (A). Orange and purple indicate high and low median-centered expression levels, respectively. Cell lines and miRNAs are clustered by similarity as determined by Euclidean distance. (C) Western blotting for abundance of epithelial markers (P-cadherin, E-cadherin, and β-catenin) and vimentin as a mesenchymal marker in a panel of KM (red text) and KE (green text) NSCLC cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a gel loading control for (D) and (E). (D) Western blot showing effects of miR-200c and miR-205 reconstitution in H460 KM cells at two concentrations of synthetic mimic oligonucleotides (10 and 50 nM) on the abundance of epithelial and mesenchymal markers. Data are representative of two independent experiments. (E) Confocal immunofluorescence micrographs of H460 cells transfected with control or miR-200c mimics. Cells are shown costained with E-cadherin (green channel) and vimentin (red channel). Scale bars, 25 μM.

  • Fig. 2 Reconstitution of specific KE-correlated miRNAs causes reduced cell viability in KM cell lines.

    (A) Alamar Blue–based cell proliferation/viability assays measuring relative viability of indicated cell lines after transfection with 10 nM synthetic miRNA mimic oligonucleotides compared to NC-transfected cells 72 hours after transfection. Data are means of three replicates ± SEM. Viability effects of miR-124 are highlighted with a red asterisk. (B) Heat map representing normalized median-centered viability data from (A). Cell lines and miRNAs are clustered by a Euclidean distance similarity metric. miR-124 is highlighted in red text.

  • Fig. 3 miR-124 reconstitution causes cytotoxic effects in KM cell lines.

    (A) Luciferase-based caspase-3 activation assays after reconstitution of indicated miRNA mimics in H460 KM cells, H322 KRAS-WT NSCLC cells, and MCF7 KRAS-WT breast cancer cells. (B) Three-dimensional (3D) Matrigel assays of A549 KM cell growth after lentiviral expression of control luciferase or primary MIR124 (pri-MIR124). Fixed cells are shown stained with phalloidin to visualize actin filaments (red signal) and 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA (blue signal). Arrow indicates an apoptotic cell with fragmented DNA and low actin content. Scale bars, 25 μm. (C) Automated image-based quantitation of colony size and number from 3D Matrigel assays shown in (B). Colony size is in arbitrary pixel units. Bars represent population means ± SEM. (D) Bright-field microscopy images of NC- or miRNA-transfected cells. Arrows indicate large intracellular endosomal, possibly vacuolar structures. Scale bars, 25 μm. Data are representative of three independent experiments.

  • Fig. 4 miR-124 reconstitution blocks autolysosome accumulation in KM cells.

    (A) Schematic overview of the mCherry-GFP-LC3 reporter. (B) Live-cell imaging of H460 KM cells expressing the mCherry-GFP-LC3 reporter and transfected with miR-124 oligonucleotide dsRNA and/or treated with 50 μM chloroquine. Arrows indicate LC3 aggregates that are GFP and mCherry double-positive, indicative of phagophore or autophagosome accumulation. Time is shown in hours. Scale bars, 25 μm. (C) Quantitation of GFP/mCherry double-positive puncta, indicating phagophores/autophagosomes (AP) using image analysis software (BioTek Gen5). (D) Quantitation of mCherry-positive autolysosomes (AL). (E) Quantitation of AL/AP ratio. Data are representative of three independent experiments.

  • Fig. 5 miR-124 reconstitution represses beclin 1 and p62 expression selectively in KM cells.

    (A) Western blot showing protein expression levels of cleaved PARP, indicative of apoptosis, and autophagy pathway components beclin 1, ATG16L1 α/β splice isoforms, p62, and LC3-I/II after transfection with miR-124 in two KRAS wild-type (WT) cell lines (MCF7 and H322) and four KM cell lines. GAPDH is a gel loading control. (B) Western blot analysis of effects of miR-124 transfection minus/plus chloroquine treatment on expression levels of p62, NBR1, and LC3-II in H460 cells. (C) Western blot showing indicated protein levels after miR-124 reconstitution in H460 KM cells compared to H358 KE cells. Cleaved (Δ) PARP indicates apoptosis. E-cadherin and vimentin indicate epithelial versus mesenchymal differentiation. (D) Western blot analysis of cleaved PARP, E-cadherin, vimentin, and p62 in H358 KE cells compared to a stable mesenchymal derivative cell line H358M after miR-124 transfection. GAPDH serves as a gel loading control for (A) to (C) and (F). (E) Quantitation of endogenous mature miR-124 levels by qPCR in H358M cells relative to parental H358 cells. **P < 0.005. (F) Schematic representation of the human p62/SQSTM1 3′UTR showing the miR-124 binding site seed sequence and point mutants (1 and 2) that disrupt the miR-124/ SQSTM1 interaction. Reporter activity of WT or mutant SQSTM1 3′UTRs after miR-124 transfection. Data are means of three replicates + SEM. ***P < 0.0005; n.s., not significant. (G) Rescue of miR-124–induced cell viability defects with exogenous GFP-tagged p62 expression in A549 and SW900 KM cell lines. Data are means of three replicates ± SEM. **P < 0.005. All data are representative of three independent experiments.

  • Fig. 6 miR-124 reconstitution induces an altered expression of NF-κB–regulated inflammatory cytokines.

    (A) Western blots showing protein expression levels of the p65 (RELA) subunit of NF-κB and TRAF6 after miR-124 reconstitution in the indicated KM cell lines. GAPDH is a gel loading control. (B) NF-κB–dependent luciferase reporter assays showing arbitrary luminescence counts after control NC or miR-124 transfection in A549 and SW900 KM cells. ***P < 0.0005, **P < 0.005. (C) Antibody-based proteomics array showing expression levels of inflammatory cytokines in conditioned media from NC or miR-124–transfected H460 KM cells. Most significantly altered cytokines are highlighted in boxes. (D) Relative quantitation of pixel density from arrays shown in (C), comparing miR-124– to NC-transfected cells. Data are representative of two independent experiments.

  • Fig. 7 A model of miR-124 activity in KRAS mutant mesenchymal (KM) cells.

    In KM cells, under basal conditions, the balance between cell survival and cell death is tightly regulated by autophagy and NF-κB signaling pathways. Upon miR-124 reconstitution in KM cells, abundance of SQSTM1/p62, BECN1, TRAF6, and RELA was suppressed, causing inhibition of autophagy flux and perturbation of NF-κB signaling. Ultimately, these coordinated miR-124 effects tip the balance in favor of cell death.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/496/eaam6291/DC1

    Fig. S1. Regulation of mesenchymal-epithelial transition by KE-associated miRNAs.

    Fig. S2. Effects of primary MIR-124 lentiviral expression on cellular growth and viability.

    Fig. S3. Live-cell imaging of autophagic flux in H460 KM cells.

    Fig. S4. BECN1 expression is suppressed by miR-124.

    Fig. S5. Repression of p62 and downstream signaling by miR-124.

    Fig. S6. Control of inflammatory cytokine expression by miR-124.

    Table S1. TLDA raw data.

    Table S2. TargetScan miR-124 predicted targets.

    Table S3. Primer and oligo sequences.

    Movie S1. Live-cell imaging of mCherry-GFP-LC3 in control H460 cells.

    Movie S2. Live-cell imaging of mCherry-GFP-LC3 in miR-124–transfected H460 cells.

    Movie S3. Live-cell imaging of mCherry-GFP-LC3 in chloroquine-treated H460 cells.

    Movie S4. Live-cell imaging of mCherry-GFP-LC3 in miR-124 + chloroquine–treated H460 cells.

  • Supplementary Materials for:

    Regulation of autophagy, NF-κB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells

    Anita K. Mehta, Kevin Hua, William Whipple, Minh-Thuy Nguyen, Ching-Ti Liu, Johannes Haybaeck, Joanne Weidhaas, Jeff Settleman, Anurag Singh*

    *Corresponding author. Email: asingh3{at}bu.edu

    This PDF file includes:

    • Fig. S1. Regulation of mesenchymal-epithelial transition by KE-associated miRNAs.
    • Fig. S2. Effects of primary MIR-124 lentiviral expression on cellular growth and viability.
    • Fig. S3. Live-cell imaging of autophagic flux in H460 KM cells.
    • Fig. S4. BECN1 expression is suppressed by miR-124.
    • Fig. S5. Repression of p62 and downstream signaling by miR-124.
    • Fig. S6. Control of inflammatory cytokine expression by miR-124.
    • Legends for tables S1 and S2
    • Table S3. Primer and oligo sequences.
    • Legends for movies S1 to S4

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.22 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). TLDA raw data.
    • Table S2 (Microsoft Excel format). TargetScan miR-124 predicted targets.
    • Movie S1 (.avi format). Live-cell imaging of mCherry-GFP-LC3 in control H460 cells.
    • Movie S2 (.avi format). Live-cell imaging of mCherry-GFP-LC3 in miR-124–transfected H460 cells.
    • Movie S3 (.avi format). Live-cell imaging of mCherry-GFP-LC3 in chloroquine-treated H460 cells.
    • Movie S4 (.avi format). Live-cell imaging of mCherry-GFP-LC3 in miR-124 + chloroquine–treated H460 cells.

    [Download Tables S1 and S2]


    Citation: A. K. Mehta, K. Hua, W. Whipple, M.-T. Nguyen, C.-T. Liu, J. Haybaeck, J. Weidhaas, J. Settleman, A. Singh, Regulation of autophagy, NF-κB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells. Sci. Signal. 10, eaam6291 (2017).

    © 2017 American Association for the Advancement of Science