Analysis of melanoma samples from the Cancer Genome Atlas
The miRanda Database was used to generate a list of miRNAs predicted to target PHB1. Data from The Cancer Genome Atlas (TCGA) were used to evaluate the expression of miR-195 and PHB1. We downloaded level 3 data of 341 matched mRNA-Seq and miRNA-Seq tumor samples, as well as one normal sample for each data set. Pearson correlation was used to calculate pairwise correlations between PHB1 and miRNAs expression. Gene expression analyses comparing melanoma samples with normal samples were performed using EdgeR .
Human melanoma cell lines SK-MEL-5, SK-MEL-19, SK-MEL-37, SK-MEL-147, UACC-62, WM35, WM793B, WM1366, WM1552C, WM1617, Lox10, MZ2Mel, and Human immortalized keratinocytes (HaCat) were maintained with DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (10,000 units/mL of penicillin and 10,000 μg/mL of streptomycin). Human melanocytes (NGM) were maintained with DMEM/F-12 medium supplemented with 20% FBS and 1% Human Melanocyte Growth Supplement (HMGS) (LifeTechnologies/Thermo Fisher Scientific, Waltham, MA, USA). HeLa cells were maintained with RPMI medium supplemented with 10% FBS and antibiotics. The sources of all cell lines used at this study are described in detail in Additional file 1: Table S1. UACC-62 and SK-MEL-5 were selected for functional assays since these lines were isolated from metastatic melanoma and are positive for the BRAF-V600E mutation . Cells were screened monthly for Mycoplasma contamination.
MicroRNAs mimics transfection
UACC-62 and SK-MEL-5 cells were transfected with microRNA mimics using Lipofectamine RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). We used miRNA mimic Syn-has-miR-195 (5′-TCCTTCATTCCACCGGAGTCTG-3′) (GE Dharmacon, Lafayette, CO USA) and ALL STARS Negative control siRNA (QIAGEN, Hilden, Germany). PHB1 expression in melanoma cells was evaluated by quantitative real time polymerase chain reaction (RT-qPCR) and western blot 48 h (24 h mimics plus 24 h of drugs) and 72 h (24 h mimics plus 48 h of drugs) after treatment, respectively.
Stable UACC-62 cells expressing PHB1 were reversely transfected with four siRNAs (25 nM) sequences targeting PHB1 (Dharmacon, ON-TARGETplus SMARTpool siRNA J-010530-05,-06,-07, and −08, Thermo Scientific) using Lipofectamine RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Negative control ON-TARGETplus Non-targeting siRNA reagent (D-001810-01-05) was obtained from Dharmacon. Endogenous and recombinant PHB1 expression were evaluated 72 h after siRNA transfections and identified by immunoblotting assay.
Plasmids construction and site-directed mutation
A 852 bp (position 82–934) fragment of PHB1 3’UTR region (PHB1–3’UTR-WT) was synthesized by GeneArt System (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and sub-cloned into the pmirGLO Dual-Luciferase miRNA Target Expression vector (Promega, Madison, WI USA) at NheI/XhoI restriction sites. Site-directed mutation was performed in order to delete miR-195 binding-site region (PHB1–3’UTR-del195–5′-…agaTGCTGCTgaa…3′) using Pfu Turbo DNA polymerase (2.5 U/μL) following the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). PHB1-ORF (819 bp) was cloned into a pENTR223 cassette in an ORFExpress System (GeneCopoeia, Rockville, MD USA) and then into a pcDNA3.1-nV5-DEST plasmid using the Gateway System (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Sanger sequencing confirmed all construct inserts.
Stable cell lines generation
UACC-62 cells stably expressing PHB1-ORF (Open Reading Frame, without 5′ and 3’UTR) or pcDNA3.1-EV (empty vector) (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were generated by transfection followed by G418 selection (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) (0.8 mg/mL). Plasmid transfections were carried out using the Lipofectamine 3000 reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). The PHB1 expression level was monitored using immunoblotting assays.
After lysis with TRIzol® reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA), total RNA was isolated from the aqueous phase upon mixing with chloroform, precipitated with isopropanol, washed with 75% ethanol and re-suspended in nuclease-free water. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Quantitative RT-PCR for PHB1 (Fwd: 5′-GTGTGGTTGGGGAATTCATGTGG-3′; Rev.: 5′-CAGGCCAAACTTGCCAATGGAC-3′), and endogenous control A-CTB (Fwd: 5′-CCTGGCACCCAGCACAAT-3′; Rev.: 5′-GGGCCGGACTCGTCATACT-3′) were carried out using SYBR Green Master Mix (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA USA). The miRNA-195 or RNU48 (endogenous control) transcripts were quantified using TaqMan Small RNA assays (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). All reactions were performed in an ABI 7500 Real Time PCR machine (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA USA) and data were acquired using the ABI SDS 2.0.1 software package and analyzed using the 2-
After collection, cells were suspended and sonicated in 2xSDS Laemmli sample buffer. A 12% SDS-PAGE gel with a 4% stacking gel was run in Tris-glycine- SDS buffer. A semi-dry transfer procedure onto a nitrocellulose membrane was carried out. After transfer, the membrane was blocked with Tris-buffered saline (TBS) with 1% Tween-20 and 5% milk. Membranes were probed with a goat polyclonal anti-Prohibitin 1 antibody (PHB1, 1:200, Santa Cruz, Santa Cruz, CA USA), mouse-V5 Tag Monoclonal antibody (V5-Tag, 1:4000, Invitrogen), mouse monoclonal anti-alpha Tubulin antibody (TUB, 1:2000, Sigma), and monoclonal anti-beta-actin antibody (ACT-B, 1:2000, Abcam, Cambridge, UK). Horseradish peroxidase (HRP)-conjugated anti-Goat IgG antibody (1:6000) was used as a secondary antibody for anti-PHB1 while HRP-conjugated goat anti-mouse IgG antibody (Pierce) was used as a secondary antibody for anti-TUB and anti-ACT-B (for both 1:4000) and for anti-V5-Tag (1:8000). Proteins were detected using the electro-chemoluminescence FluorChem R System (Protein Simple, San Jose, CA, USA).
Cell proliferation assay
Cell proliferation assay was conducted using UACC-62 and SK-MEL-5 cell lines seeded in 96-well plates (3 × 103 cells per well). Cells were reverse transfected (RNAiMax) with miRNA-195/miRNA-control mimics (10 nM). After 24 h, cells were treated with cisplatin (2.5, 5.0 and 10 μM, SIGMA, Darmstadt, Germany), temozolomide (50, 250 and 450 μM, SIGMA, Darmstadt, Germany) or DMSO 0.1% as vehicle. Forty-eight hours after treatment, the nuclear counting per mm2 (%) of treated cells was compared to the non-treated cells (IncuCyte, Essen BioScience, Ann Arbor, MI, USA). For miR-195-PHB1 antagonism studies, two clones of the UACC-62 cell line overexpressing either ORF-PHB1 or pcDNA3.1-EV were used. MicroRNA-195 or miRNA-control was transfected into stable each cell line. Nuclear counting per mm2 was carried out daily for five days after transfection using IncuCyte software and viability of control (%) was calculated.
Cell death and cell cycle analysis
UACC-62 and SK-MEL-5 cells were seeded at 2 × 105 cells per well in a 12 multiwell plate. Cells were reverse transfected (RNAiMax) with miRNA-195/miRNA-control mimics (10 nM). After 24 h, cells were treated with cisplatin (2.5 and 10 μM), temozolomide (50 and 250 μM) or DMSO 0.1%. After 48 h, cells were trypsinized, fixed in 70% ethanol and kept at −20 °C until analysis by flow cytometry (Attune® Acoustic Focusing Cytometer, Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Cell death and cell cycle analysis were performed by propidium iodide (PI) staining. PI incorporates stoichiometrically to DNA, allowing relative quantitation of DNA content. Cell death analysis, indicated as hypodiploid cells (Sub-G1) and cell cycle distribution (G0/G1, S, and G2/M) analysis were performed using the FlowJo v10 Cytometric Software algorithm (FlowJo LLC, Ashland, Oregon, USA). The percentage of cell death was expressed in bar graphs (GraphPad, La Jolla, CA). Cell cycle distribution profiles were plotted in a chart.
Caspase 3/7 apoptosis assay
A caspase 3/7 activity-based assay was performed for apoptosis quantification. UACC-62 and SK-MEL-5 cells were seeded in 96 well plates and reverse transfected with either miR-195 or miRNA-control (10 nM). After 24 h, cells were exposed to cisplatin or temozolomide (2.5 and 50 μM, respectively). After 48 h, the apoptosis index was monitored in the supernatant using the Caspase-Glo 3/7 Assay Reagent according to manufacturer’s instructions (Promega, Madison, WI, USA). Luciferase measurements were performed with the SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
Dual-GLO luciferase assay
For the luciferase assays, 8 × 103 HeLa cells were plated 24 h prior to plasmid transfection in a 96-well plate in triplicate. 10 ng of each pmiR-GLO-3′-UTR-PHB1 or pmiR-GLO-PHB1–3’UTR-del195 reporter vector were mixed with 500 nM of each miRNA-195 or miRNA-control in 25 μL OptiMEM (Invitrogen/Thermo Fisher Scientific, Waltham, MA USA). A 0.5 μL aliquot of Lipofectamine 2000 transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA USA) was added in 25 μL OptiMEM. Mixes were combined and after formation of the nucleic acid:lipid complex, the transfection solution was overlaid onto the previously plated HeLa cells. HeLa cells were selected for luciferase assays based on their high transfection efficiency and reproducibility according to our previous experience [26, 27]. After incubation for 48 h, a HeLa cell extract was prepared using the Reporter Lysis Buffer (Promega, Madison, WI, USA). A 50 μL amount of Luciferase Assay Reagent (Promega, Madison, WI USA) was added to 10 μL of cell lysate and luminescence was measured with a GloMax-Multi + Microplate Multimode Reader (Promega, Madison, WI, USA). Data were normalized by Firefly/Renilla luciferase activity.
Statistical analyses were conducted using GraphPad Prism Software v6.01 (GraphPad, La Jolla, CA). The difference between two groups were analyzed by the unpaired t test. The differences between three or more groups were analyzed by ANOVA with Tukey’s multiple comparisons test. A value of P ≤ 0.05 was considered to be statistically significant.