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. 2016 Jul 5;7(27):42274-42287.
doi: 10.18632/onco_target.9786.

MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis

Min Cheng  1   2 , Lei Liu  1   2 , Yuanzhi Lao  3 , Weijie Liao  1   2 , Meijian Liao  1   2 , Xuan Luo  2   4 , Jiangbin Wu  2 , Weidong Xie  2 , Yaou Zhang  2   5 , Naihan Xu  2   5
Affiliations

MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis

Min Cheng et al. Onco_target. .

Abstract

Damage to mitochondria often results in the activation of both mitophagy and mitochondrial apoptosis. The elimination of dysfunctional mitochondria is necessary for mitochondrial quality maintenance and efficient energy supply. Here we report that miR-181a is a novel inhibitor of mitophagy. miR-181a is downregulated by mitochondrial uncouplers in human neuroblastoma SH-SY5Y cells. Overexpression of miR-181a inhibits mitochondrial uncoupling agents-induced mitophagy by inhibiting the degradation of mitochondrial proteins without affecting global autophagy. Knock down of endogenous miR-181a accelerates the autophagic degradation of damaged mitochondria. miR-181a directly _targets Parkin E3 ubiquitin ligase and partially blocks the colocalization of mitochondria and autophagosomes/lysosomes. Re-expression of exogenous Parkin restores the inhibitory effect of miR-181a on mitophagy. Furthermore, miR-181a increases the sensitivity of neuroblastoma cells to mitochondrial uncoupler-induced apoptosis, whereas miR-181a antagomir prevents cell death. Because mitophagy defects are associated with a variety of human disorders, these findings indicate an important link between microRNA and Parkin-mediated mitophagy and highlights a potential therapeutic strategy for human diseases.

Keywords: apoptosis; microRNA; mitochondria; mitophagy; parkin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. miR-181a is downregulated by mitochondrial uncouplers
A. Analysis of miR-181a expression in human neuroblastoma SH-SY5Y cells. Cells were treated with 10 μM FCCP or CCCP for indicated time pointes. Data shown are from five independent experiments, *p<0.05. B. GFP-LC3 puncta formation assay in SH-SY5Y cells overexpressing miR-181a or negative control miRNA (NC). Cells were treated with 10 μM FCCP or CCCP for 6 h. The distribution of GFP-LC3 was captured by confocal microscope. Scale bar, 10 μm. C. Quantification the number of GFP-LC3 puncta in the cells (n = 100 cells from three independent experiments).
Figure 2
Figure 2. miR-181a inhibits mitochondrial uncoupler-induced mitophagy
A. The relative expression of miR-181a in SH-SY5Y cells transfected with miR-181a mimics or the negative control (NC). B. SH-SY5Y cells transfected with miR-181a or NC were treated with 10 μM FCCP or C. CCCP for 12 h. Samples were collected for western blot to analyze the expression of TIM23, COXIV, C-III core, MFN1, LC3, p62, and Actin. Densitometric ratios of mitochondrial marker proteins are quantified by Image J software. D. SH-SY5Y cells transfected with NC or miR-181a were exposed to FCCP (10 μM) and HCQ (20 μM) for 12 h. Western blot was performed to analyze the status of TIM23, LC3 and Actin. E. Image J densitometric analysis of the TIM23/Actin ratios from immunoblots is shown (data from three independent experiments, *p<0.05). F. Mitochondrial content after 12 h of treatment with 10 uM FCCP was assessed by flow cytometry for NAO. G. Quantification of mitochondrial DNA copy number in NC or miR-181a transfected cells treated with FCCP (data from three independent experiments, **p<0.01).
Figure 3
Figure 3. miR-181a partially blocks colocalization of mitochondria with autophagosomes/lysosomes
A. SH-SY5Y cells overexpressing miR-181a or NC were co-transfected with GFP-LC3 and Mito-RFP for 24 h, followed by incubation with 10 μM FCCP for 6 h. The distribution of GFP-LC3 and Mito-FRP was visualized by confocal microscopy. Scale bar, 10 μm. B. Quantitative analysis of cells that contain fragmented mitochondria-localized LC3 puncta (values represent mean number of puncta counts per cell, n=100 cells from three independent experiments). C. Representative confocal images of MitoTracker Green FM (MTR green) and LysoTracker Red (LTR) in SH-SY5Y cells treated with or without FCCP for 6 h. Scale bar, 10 μm. D. Quantitative analysis of cells that contain fragmented mitochondria-localized lysosomes (n=100 from three independent experiments).
Figure 4
Figure 4. Inhibition of endogenous miR-181a promotes mitophagy
A. The relative expression of miR-181a (compared with U6) in SH-SY5Y cells transfected with miR-181a inhibitor (In-181a) or the negative control (In-NC). B. SH-SY5Y cells transfected with In-181a or In-NC were treated with 10 μM FCCP or C. CCCP for 12 h. Samples were collected for western blot to analyze the expression of mitochondrial marker proteins, LC3, p62, and Actin. Densitometric ratios of TIM23/Actin are quantified by Image J software. D. SH-SY5Y cells transfected with In-NC or In-181a were exposed to FCCP (10 μM) and HCQ (20 μM) for 12 h. Western blot was performed to analyze the status of TIM23, LC3 and Actin. E. Image J densitometric analysis of the TIM23/Actin ratios from immunoblots is shown (data from three independent experiments, *p<0.05).
Figure 5
Figure 5. miR-181a directly _targets E3 ubiquitin ligase Parkin
A. Relative Parkin mRNA levels (compared with Actin) in SH-SY5Y cells transfected with miR-181a mimics or inhibitors were analyzed by quantitative RT-PCR. Data are represented as means±s.d. from three independent experiments, and analyzed using the student t test (**p<0.01). B. SH-SY5Y and A172 cells were transfected with NC, miR-181a, In-NC, In-181a, respectively. At 48 h after transfection, cell lysates were subjected to western blot analysis for Parkin and Actin. C. Diagram of the predicted miR-181a binding sites in Parkin 3′UTR. Luciferase reporter constructs containing WT or Mut Parkin 3′UTR were co-transfected with NC or miR-181a. Relative luciferase activity was normalized to NC. Data shown are means±s.d. from three independent experiments, **p<0.01.
Figure 6
Figure 6. Silencing endogenous Parkin inhibits mitophagy
A. SH-SY5Y cells were transfected with control or Parkin siRNA for 36 h. Cells were treated with 10 μM FCCP for 12 h. Sample were collected for western blot to analyze the expression of TIM23, MFN1, Parkin, C-III core 1, p62, LC3 and Actin. B. SH-SY5Y cells transfected with control or Parkin siRNA were exposed to FCCP and HCQ for 12 h. Western blot was performed to analyze the status of TIM23, LC3 and Actin. C. Image J densitometric analysis of the TIM23/Actin ratios from immunoblots is shown (data from three independent experiments, *p<0.05, **p<0.01). D. SH-SY5Y cells transfected with miR-181a, Parkin siRNA, In-181a, or the controls were treated with 10 μM FCCP for 12 h. Representative confocal images of TMRE and MitoTracker Green FM (MTR) are shown. Scale bar, 10 μm. E. Quantification of TMRE signals in cells before and after FCCP treatment. Data shown are means±s.d. from three independent experiments, *p<0.05, **p<0.01. F. The morphology of mitochondria was visualized by MitoTracker Green FM. The number of cells show disrupted mitochondria was quantified. All data are expressed as means±s.d., *p<0.05.
Figure 7
Figure 7. miR-181a sensitizes neuroblastoma cells to apoptosis induced by mitochondrial uncouplers
A. Overexpression of Parkin restores the inhibitory effect of miR-181a on mitophagy. SH-SY5Y cells were transfected with miR-181a and Parkin plasmid as indicated. Cells were then cultured in the presence or absence of FCCP (10 μM) or CCCP (10 μM) for 12 h. Samples were collected for western blot to analyze the protein expression of TIM23, Parkin, MFN1 and Actin. B. SH-SY5Y cells transfected with miR-181a or NC were treated with 25 μM FCCP or CCCP for 24 h. Western blot was performed to analyze the status of cleaved caspase-3, cleaved PARP and Tubulin. C. Densitomeric analysis of cleaved caspase-3/Tubulin and cleaved PARP/Tubulin protein ratios. D. Flow cytometry analysis of sub G1 population in SH-SY5Y cells treated with FCCP or CCCP for 24 h. Data shown are means±s.d. from three independent experiments, *p<0.05.
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