Phosphate Buffer Saline (PBS), Hank’s balanced salt solution (HBSS), DMEM (Dulbecco’s Modified Eagle’s Medium) culture medium, culture supplements, and trypsin-EDTA (ethylenediaminetetraacetic acid) were obtained from Euroclone (Milan, Italy). LHC-8 culture medium was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The fetal bovine serum was obtained from Hyclone (Logan, UT, USA). The bovine serum albumin (BSA), collagen, epidermal growth factor (EGF), ethylene-diamine-tetra-acetic acid (EDTA), fibronectin, hydrogen peroxide (H2O2), phosphoethanolamine, propidium iodide (PI), RNAse, trypan blue solution (0.4%) and Triton X-100 were obtained from SIGMA-Aldrich (St Louis, MO, USA). Ethanol was purchased from CARLO ERBA Reagents (Milan, Italy).
Preparation of the hazelnut liquid extract (HZN)
Preparation of the ethanolic extract of hazelnut (Corylus avellana L., cultivar Tonda Gentile Romana, Coopernocciola srl, Vico Matrino, VT, Italy) was carried out as previously described . Briefly, 20 grams of raw materials were homogenized, and added to 60% aqueous ethanol solution at liquid:solid ratio of 1:5 (v/w). After 1h of continuous stirring, the extraction process was carried out in closed bottle at room temperature (20-22 ºC), in dark conditions, for 30 days. The hazelnut suspension was mixed by hand each three days. At the end of the maceration procedure, the hazelnut liquid extract (HZN) was collected, passed through a filter of 0.2μm, aliquoted and stored at -80ºC for all experiments. The complete metabolomic characterization of the HZN ethanolic liquid extract used in this study was performed by liquid chromatography-high resolution mass spectrometry, as previously detailed .
Cell cultures and treatments
HepG2 human hepatocellular carcinoma cells (RRID: CVCL_0027) were purchased from the European Collection of Cell Cultures (ECACC, Sigma-Aldrich), maintained as sub-confluent monolayers in DMEM, with 10% heat-inactivated foetal bovine serum, 2mM glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin (10,000 U/ml), at 37°C in a 5% CO2 atmosphere in air.
THLE-2 cells (RRID: CVCL_3803), derived from SV40-immortalized normal human liver cells, were purchased from the American Type Culture Collection, ATCC (Manassas, VA, USA). They were cultured in LHC-9 medium, supplemented with 70 ng/ml phosphoethanolamine, 5 ng/ml EGF, 10% fetal bovine serum (Hyclone) and antibiotics as above. Flasks and dishes for THLE-2 cultures and experiments were pre-coated with collagen (2.9 mg/ml), fibronectin (1 mg/ml) and BSA (1 mg/ml), according to ATCC guidelines.
In all the experiments, cells were seeded in 35mm Petri-dishes at 2x105 cells/dish. Twenty-four hours after plating, cells were treated with either the Hazelnut (HZN) ethanolic extract or the corresponding aqueous ethanol solution, as previously detailed [27-29]. The solutions were freshly prepared before each experiment in culture media.
The effect on cell proliferation and viability was assessed by counting viable cells at the hemocytometer, following trypan blue staining (0.4% solution). For inducing oxidative stress, THLE-2 cells were administered with H2O2 (doses ranging from 100 to 2000 mM) for 24 hours. In HZN-H2O2 combination experiments, cells were washed twice in PBS at the end of the 72 h HZN treatment, before adding hydrogen peroxide.
RNA extraction, reverse transcription and gene expression analysis
Total RNA was extracted by Trizol® (Invitrogen, Thermo Fisher Scientific) followed by spin-column elution, also including a DNAse I digestion step (Direct-zolTM RNA miniPrep, Zymoresearch, Irvine, CA, USA), as previously described . The amount and purity of the extracted RNA was evaluated by fiber optic spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, Wilmington, DE, USA) calculating the 230/260 and 260/280 absorbance ratios.
TaqMan® Reverse Transcription Reagent (Applied Biosystems, Thermo Fisher Scientific) was used to perform retro-transcription of total RNA (200 ng) with random primers, according to manufactures’ indications. Analysis of the Pri-miR-34b/c was carried out as previously described , with 1 μL of cDNA using SYBR Green master mix (Applied Biosystems) and analyzed on an Eco™ Real-Time PCR System (Illumina, San Diego, CA, USA). All reactions were run in quadruplicate, and the relative abundance of the specific mRNA levels was normalized to the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) expression, applying the 2-ΔΔCt method. PCR primers were designed by NCBI-Primer Blast free software (https://www.ncbi.nlm.nih.gov/tools/primerblast), according to gene sequences available in the UCSC database (https://genome.ucsc.edu), and selected to amplify an exon-intron-exon region (≤ 200bp) to exclude genomic contamination. PCR primers were synthesized by Eurofins Scientific (Luxembourg). Sequences were as follows: GAPDH forward (5’-GCACCGTCAAGGCTGAGAAC-3’); GAPDH reverse (5’-GAGGGATCTCGCTCCTGGA-3’); pri-miR34 forward (5’-GCTCTTTGTCCCTCCTGCTAGA-3’); pri-miR34 reverse (5’-GTGGGCGGTCCCTGAAG-3’).
Mature microRNA expression analysis
The analysis of mature miRNA-34b and miRMA-34c expression was carried out on total RNA, as previously described . Ten nanograms were retro-transcribed by the miRcury LNA universal RT microRNA kit (Exiqon, QIAGEN, Hilden, Germany); cDNA was diluted 1:80 and amplified by the miRcury LNA Sybr green master mix and miR-specific LNAPCR primer sets (Exiqon), according to the manufacturer’s instructions. All reactions were run in quadruplicate and the relative abundance of each specific microRNA was normalized to RNU1A1 small nucleolar RNAs, by applying the 2−ΔΔCt method.
Flow cytometric analysis
A FACScan flow cytometer (Becton Dickinson, Bedford, MA, USA) equipped with a 488 nm argon laser, was used for all flow cytometric analyses.
The evaluation of the DNA content for the assessment of the percentage of the apoptotic cells (sub-G1 index) was carried out as previously described . Adherent cells were harvested by trypsinization and collected with floating ones; the pool was washed twice in PBS, then fixed in ice-cold ethanol 70% (1×106 cells/ml) over-night. An aliquot of the suspension (at least 5×105 cells) was then washed twice in PBS, and stained with PI (50 μg/ml) in a mix containing RNAse A (50 μg/ml), Triton X-100 (0.1%), EDTA (0.1 mM) in PBS, in the dark, for 60 min at room temperature, then immediately analyzed. The evaluation of sub-G1 DNA content was performed by the FlowJo software®.
To quantify oxidative stress, cells were stained with the 2′,7′-dichlorofluorescin diacetate (H2DCF-DA, Molecular Probes, Thermo Fisher Scientific), and analyzed as previously described . Cells were quickly scraped on ice, washed twice in cold PBS, and re-suspended in 5 μM H2DCF-DA (30′, 37 °C in the dark, in HBSS). After a final wash in PBS, cells were immediately transferred into a tube on ice for flow cytofluorometric analysis. For each measure, a minimum of 20,000 events were evaluated. The mean fluorescence intensity (MFI) was measured as the mean fluorescence value in the channel of the H2DCF-DA-labeled cells minus the mean fluorescence value of the unstained cells.
The variations of the samples values are reported as Mean ± S.D., calculated in N = 3 independent experiments. The statistical differences were analyzed trough the KailedaGraph software (Synergy Software, Reading PA, USA) by applying the one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. P values <0.05 were considered statistically significant and indicated as follows: *P <0.05.