Mouse models
Animal housing and all the experimental procedures were authorized by the Belgian ethical committee (Protocol number: 2166). All mice used were bred in the NOD SCID background. Mice were housed 4 per cage in a temperature (22–24 °C) controlled colony room, maintained on a 12-h light/dark cycle (07:00 to 19:00 light on), with standard food and water provided ad libitum and environmental enrichments.
For tumor experiments, we used 6-week-old females per group, and we repeated each experiment twice. The allocation of mice in different group was random. During experiments, mice were monitored twice per day. Behavior, weight, furrow, and tumor size were controlled every day. The total number of mice analyzed for each experiment is detailed in the figure legends. All in vivo experiments were approved by the local Animal Ethic Commission (File number 2166, “Commission d’éthique de l’utilisation des animaux de l’Université de Liège”).
Cell culture
All cells were grown at 37 °C in a 5% CO2 humid atmosphere. MDA-MB-231 breast adenocarcinoma cells (ATCC-HTB26; sex: F), MCF7, HS578T and T47D (kindly p-rovided by Dr. Gilles, University of Liège) were cultured in DMEM containing 1 g/L glucose, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.
Generation of MDA-MB-231 OPA1
−/− cells
MDA-MB-231 OPA1−/− cells were generated by CRISPR/Cas9. Using the CRISPR Design Tool at crispr.mit.edu we designed the following guide RNAs targeting Exon 2 of OPA1:
Oligos were annealed using T4 Polynucleotide kinase, ligated into the Cas9 from S. pyogenes with 2A-EGFP plasmid (PX458, Addgene Plasmid #48138) cutting at a BbsI restriction enzyme site. XL10-Gold ultracompetent bacteria were transformed and grown under ampicillin selection. Resulting colonies were picked and the insertion of the guide RNA’s were confirmed by sequencing. DNA was purified using a QIAprep Spin Minprep Kit (Qiagen) according to manufacturer’s instructions. The successful insertion of the guide RNAs into the vector was confirmed by the loss the restriction enzyme site for BbsI. The generated plasmids were transfected into MB-MDA-231 cells using Lipofectamine 3000 according to the manufacturer’s instructions. 48 h post-transfection, the GFP+ cells were single sorted by flow cytometry (FACS Aria, BD Biosciences). Single clones in 96-well plates were cultured and expanded in complete growth media and OPA1 levels were evaluated by western blot.
Design of the synthetic miRNA
The miRNA mimics miR-148a-3p, miR-148b-3p, miR-152-3p and cel-miR-67 (control) are double-stranded RNAs designed using the method of Betancur et al. [30]. The mature miRNA strand was modified by the addition of phosphorylation at the 5′ end and the carrier strand was the complementary RNA sequence, carrying a two base 3′ overhang with mutations near the 3′ end to thermodynamically destabilize the strand and induce faster degradation. Oligonucleotides were purchased from Eurogentec.
MiRNA and siRNA transfection
Cells (18 × 105 per well in a 6-wells plate; 8 × 105 per well in a 48-wells plate and 8 × 104 per well in a 96-wells plate) were transfected with the following siRNAs: a non-relevant human sequence (UNREL) (AM4635 Ambion, 20 nM), OPA1 (144,409 Invitrogen, 20 nM), OPA1 siRNA2 (36,409, Invitrogen 20 nM), MFN1 (50 nM) and MFN2 (50 nM) using Dharmafect-4 (Dharmacon) according to manufacturer’s instructions. All transfection experiments described in this paper were carried out with MFN1 and MFN2 siRNA1. Similar results were obtained with MFN1 and MFN2 siRNA 2 (data not shown). Functional assays on transfected cells and evaluation of transfection efficiency were performed after 72 h. Cells were trypsinized, stained with trypan blue and living cells were seeded for the different functional assays.
For miRNAs transfection, pre-miR-148a, 148b and 152 and anti-miR-148a, 148b and 152 and the non-relevant human sequences (P-UNREL for the pre-miR-control and A-UNREL for the anti-miR-control) were used at a concentration of 10 nM following the same protocol as siRNAs transfection.
Plasmid transfection
30 × 105 cells were seeded on 6-well plate and the transfections were performed using Lipofectamine 3000 according to the manufacturer’s instructions. For transient overexpression of Opa1, MDA-MB-231 were transfected with pMSCV-Opa1 [21] and the assays performed after 48 h.
Cell proliferation assay
Cells in a 96-well plate were transfected in 100 μl DMEM 1 g/L glucose for 72 h. Medium was changed with DMEM supplemented with 10% FCS for 24 h to induce proliferation. The thymidine analogue 5-bromo-2-deoxyuridine (BrdU) was added and incubated for 16 h. BrdU incorporation was measured with the Cell Proliferation ELISA BrdU chemiluminescence kit (Roche Applied Science) according to the manufacturer’s protocol. Where indicated, cells were treated with MYLS22 (50 μM) or DMSO when the complete DMEM medium was added.
Scratch wound migration assay
Cells in 48-wells plates were transfected in 800 μL of DMEM 1 g/L glucose for 72 h and the head of a 200 μL tip was used to perform a wound. Migration of cells into the wound was measured 6 h later. The percentage of coverage was calculated using the following formula:
$$\%\mathrm{Coverage}\:=\:\lbrack({\mathrm{Width}}_{\mathrm T0}-{\mathrm{Width}}_{\mathrm T6})/{\mathrm{Width}}_{\mathrm T0}\rbrack\ast100$$
Where indicated, cells were treated with MYLS22 (50 μM) or DMSO when the scratch was performed.
Boyden chamber assay
Cells were seeded on the upper wells of 8-μm Boyden chambers (24-wells Transwell, Costar Corp) and incubated for 16 h in in 300 μl DMEM without FCS. To induce migration, the lower chamber was filled with 600 μl of DMEM supplemented with 10% FCS. Cells were allowed to migrate for 6 h at 37 °C. The membrane insert was removed and flipped so that the side towards the lower chamber faced the operator. Cells were fixed for 10 min in MeOH (100% V/V, ice cold) and stained with 4% Giemsa and the insert was mounted on microscope slides. Cells were imaged using an Olympus CKX41 microscope (Olympus Life Science) and counted using ImageJ (NIH). Where indicated, the media in the upper chamber was supplemented with MYLS22 (50 μM) or DMSO when migration was induced.
Cell adhesion assay
Cells (3 × 104) were seeded on Fibronectin- or Gelatin-coated 96-wells plate for 1 h, washed with PBS and fixed for 10 min in MeOH (100% V/V, ice cold). Cells were then stained with Crystal violet solubilized in acetic acid for 15 min and washed three times. Crystal violet absorbance was measured at 520 nM. Where indicated, cells were pretreated with MYLS22 (50 μM) or DMSO 24 h before the assay.
Invasion assay
Spheroids were prepared as previously described [31]. Homospheroids composed of transfected MDA-MB-231 were allowed to form in 96-well suspension plates for 48 h. Spheroids were then collected and seeded for 24 h in a 3D collagen matrix with culture medium and methylcellulose. To quantify invasion of collagen matrix by MDA-MB-231 cells, wells were imaged using an Olympus CKX41 microscope (Olympus Life Science) and the areas of invasion and of the spheroids was calculated using ImageJ (NIH). The invasion rate was determined as the ratio of the area of invasion over the area of the spheroid.
Apoptosis assay
WT and MDA-MB-231 OPA1−/− cells (1 × 105) were grown in 6-wells plates. After 24 h cells were stained with Annexin-V-FICT and propidium iodide (PI), according to manufacturer’s protocol (eBioscience™). The rate of cell death, expressed as percentage of the Annexin-V-positive events in the total population, was measured by flow cytometry (FACS Calibur, BD Biosciences). Apoptosis was determined as described above in transfected MDA-MB-231 cells that were transferred to the 6 wells plates 48 h after the transfection.
Electron microscopy
Cells grown in 24-wells plates were fixed for 1 h at 4 °C with freshly prepared 2.5% (V/V) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4. After washing with 0.1 M sodium cacodylate, cells were post-fixed in 1% OsO4, 1.5% K4Fe(CN)6 in 0.1 M sodium cacodylate pH 7.4, stained with 0.5% uranyl acetate, dehydrated in ethanol and embedded in Embed 812. Thin sections were imaged on a Tecnai-12 electron microscope (Philips-FEI) equipped with a Veleta (Olympus Imaging System) digital camera at the BioImaging Facility of the Dept. of Biology (University of Padua).
Seahorse
Oxygen consumption rate (OCR) was measured with the Agilent Seahorse XFe24 Analyzers. WT, MDA-MB-231 OPA1−/−and transfected cells (3 × 106) in complete DMEM were seeded in XF24 cell culture microplates, resulting in 60–70% confluency. After 24 h, the medium was replaced with XF medium (DMEM 1 g/L glucose, 0.58 g/L Glutamine, 1 mM Sodium Pyruvate, 0.015 g/L Phenol Red, pH 7.2) and plates were transferred to a 37 °C incubator not supplemented with CO2 for 1 h to equilibrate temperature and pH. After three measurements of basal OCR, 70 μl of solutions containing oligomycin, FCCP or rotenone and antimycin A were sequentially added to each well to reach final concentrations of 1 μM oligomycin, 1 μM FCCP and 2 μM for rotenone and antimycin A. Following each compound injection, three measurements were acquired. OCR is expressed as pmol O2 per min.
Mitochondrial membrane potential measurement
Cells were incubated with tetramethyl rhodamine methyl ester (TMRM, Invitrogen) were incubated at 37 °C for 30 min with 1 nM TMRM in the presence of 1 μg/ml cyclosporine H (CsH), a P-glycoprotein inhibitor. Where indicated, 2 μg/ml oligomycin and 2 μM FCCP were added for an additional 30 min. TMRM fluorescence was measured by flow cytometry (FACS Aria, BD Biosciences).
ATP measurement
ATP levels in MDA-MB-231 cells were measured 72 h after transfection using ATPlite kit (Perkin Elmer) according to the manufacturer’s protocol.
miRNAs extraction, cDNA production and gene expression
Total RNA was extracted using the miRNeasy kit (Qiagen) following manufacturer’s protocol. For Poly(A) tailing and reverse-transcription of miRNAs (BioLabs), 20 ng RNA was reverse transcribed into cDNA. The expression levels of miRNAs were analyzed using SYBR® Green I kit (Eurogentec) by 7900 Real-Time PCR System (Applied Biosystems) and the results were obtained with the 2-ΔCt method [32]. The miRNA levels were normalized to RNU44 and RNU48.
miRNA PCR array
MicroRNAs expression was evaluated using a Breast Cancer Focus microRNA PCR Panel, 96-well (Qiagen). Each plate contains 84 lyophilized LNA miRNA primer sets focusing on cancer-relevant human miRNAs, 3 reference genes (miRNAs and small nuclear RNAs, snRNAs), 3 inter-plate calibrators and 5 RNA spike-ins. According to manufacturer’s protocol,10 μl of the master mix (5 μl of cDNA previously synthetized, 500 μl SYBR Green, 50 μl of ROX Reference dye and 445 μl of nuclease-free water) was added in each well. qPCR was performed using the 7900 Real-Time PCR System (Applied Biosystems) according to the following conditions: 2 min at 95 °C then 40 cycles at 95 °C for 10 s and 56 °C for 1 min. The Qiagen tool Geneglob was used to analyze the data. The relative miRNA levels were normalized to global mean.
MiRpaths analysis method
The DIANA-miRPath v3.0 analysis was used to determine the molecular pathways controlled by miRNAs 148a, 148b and 152 annotated on the Kyoto Encyclopedia of Genes and Genomes (KEGG), using the following default parameters: experimentally supported interactions from DIANA TarBase v.7.0; a p-value threshold of 0.05; and a microT threshold of 0.8. To reduce the number of false-positive miRNA targets, we applied a false discovery rate (FDR) correction to selected KEGG pathways. The algorithm used in this analysis was a one-tailed Fisher exact test.
Analysis of gene expression by qRT-PCR
RNAs were extracted with the miRNeasy kit (Qiagen) according to manufacturer’s protocol. cDNA synthesis was performed with 1 μg total RNA and the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s instructions. Resulting cDNAs (20 ng) were used for quantitative real-time PCR using the SYBR green method (Roche Applied Sciences). Thermal cycling was performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). For all reactions, no template controls were run, and random RNA preparations were also subjected to sham reverse transcription to check for the absence of genomic DNA amplification. Quantitative real-time PCR was performed with SYBR green method (Bioline and ThermoFisher Scientific). Thermal cycling was performed on an Applied Biosystem 7900 HT detection system and a Stratagene MX3005P multiplex QPCR system (Applied Biosystems and Stratagene).
The relative transcript level of each gene was normalized to the housekeeping genes cyclophilin-A (PPIA), beta-2 microglobulin (B2M) and/or GAPDH. Primers were designed using Primer Express software and selected to span exon-exon junctions to avoid detection of genomic DNA (primer sequences are provided in supplementary Methods). Quantification of mRNA levels was calculated with the 2-ΔCt method.
Western blotting
Cells were lysed in lysis buffer and heated at 95 °C for 10 min. Equal amounts of protein were resolved by 8% SDS-PAGE (Biorad) and transferred to polyvinylidene fluoride membranes (PVDF) according to the manufacturer’s protocol. Membranes were blocked for 1 h at room temperature with 5% BSA (Sigma Aldrich) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and probed overnight at 4 °C with the indicated primary antibodies. After 3 washes with TBS-T, the appropriate secondary antibody at a 1:5000 dilution was added for 1 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence ECL kit (Pierce).
Immunofluorescence
Cells (3 × 105) were cultured on coverslips coated with gelatin (0.2%), washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, blocked, permeabilized with PBS containing 5%BSA and 0.5% saponin, and incubated overnight in PBS supplemented with 1% BSA and 0.1% saponin (PBS-BS, Sigma Aldrich) containing the indicated primary antibodies. After 3 washes with PBS cells were incubated with the appropriate secondary antibodies conjugated to Alexa Fluor 488 and 568 (Thermo Fisher Scientific) dissolved in PBS-BS for 1 h at room temperature. Samples were washed 3 times with PBS and coverslips were mounted on microscope slides using the DAPI containing ProlongFade (Invitrogen) mounting solution. Slides were placed on the stage of a LSM700 (Zeiss) confocal microscopy equipped with a 63X, Zeiss Plan-Apochromat 63x/1.4 Oil objective and excited using the appropriate laser line. Images were acquired using a 1048 × 1048 resolution with the ZEN software (Zeiss).
For immunofluorescence on tissues, 5 μm thick cryostat sections were fixed in acetone at − 20 °C and in 100% methanol at 4 °C and then incubated with rat monoclonal anti-PECAM-1 for 1 h at room temperature. Slides were washed and incubated with Alexa 488 anti-rat for 1 h at 4 °C. After three washes, slides were covered with coverslips in mounting medium (Victor Laboratories) and analyzed using a Leica DMI4000 epifluorescence microscope (equipped with 10X/0.25, 20X/0.35NA, 40X/0.6NA and 63X/1.25NA objectives).
Orthotopic MDA-MB-231 adenocarcinoma implants
Subconfluent MDA-MB-231 OPA1−/−:OPA1 or MDA-MB-231 OPA1−/− cells were trypsinized, washed and resuspended in PBS. The MDA-MB-231 cell suspensions (5 × 104 cells in 100 μl matrigel or 1 × 105 cells in 100 μl PBS) were injected in the fourth mammary gland of each mouse. For MYLS22 treatment, a solution containing 10 mg/kg MYLS22 resuspended in corn oil was injected peritumorally from day 21 (when tumor size reached 50mm3) every second day. Thirty-six days after tumor cells injection, mice were euthanized, and their tumors harvested.
Tumor growth of MDA-MB-231 cells was assessed by measuring the length and width of each tumor every day. Tumor volume was calculated using the following formula:
$$\mathrm V=\mathrm l\;\ast\mathrm w^2\;\times\;0.5$$
Where V: volume; w: width; l: length.
Quantification and statistical analysis
Data representation and statistical analysis
Data are displayed as dot plots of at least 3 independent experiments. Plots include each datapoint, mean and SEM. In case of tumor growth curve, average ± SEM is shown. Normal distribution of populations at the 0.05 level was calculated using the Shapiro-Wilk normality test. Statistical significance was calculated by a one-way ANOVA with Tukey’s post-test using OriginPro (Microcal). All P values and n are reported in the figure legends. Results are considered significant when p < 0.05.