Design of modified small oligoribonucleotides targeting SIRT1-NAT
To knockdown SIRT1-NAT, we designed an siRNA and 2 antagoNATs. The 17-19 nucleotide antagoNATs contained phosphorothioate modification plus 2′-O-methyl RNA nucleotides. In addition, control siRNA and anatoNATs were designed. Sequences of the antagoNATs and the siRNA are listed in Supplementary Table 1.
Cell culture
Isolated MSCs from human adipose tissue24 were cultured in Dulbecco’s modified Eagle’s medium (DMDM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Nantong, China) and incubated at 37° C and 5% CO2. MSCs were characterized by flow cytometric analysis for the expression of the typical markers CD90, CD73, and CD105 and absence of the hematopoietic markers CD14, CD45, CD3424. Every 2-3 days, upon 80% confluency, the cells were subcultured at a ratio of 1:3. MSCs at passages 2 and 7 were used in all experiments.
Small RNA transfection
5 × 104 MSCs were seeded into each well of 12-well plates. After overnight incubation, the cells were transfected with siRNA or antagoNATs using Lipofectamine TM 2000 reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 48 hours post-transfection, cells were collected for further experiments.
RNA extraction and Real-time qPCR
Total RNA was extracted from the MSCs using Hybrid-RTM RNA Extraction Kit (GeneAll, Seoul, South Korea), followed by treatment with DNase I (RNase-free; Invitrogen, Carlsbad, CA, USA) at 37°C for 15 min to eliminate DNA contamination. Reverse transcription reactions were performed to synthesize cDNA using RevertAid M-MuLV Reverse Transcriptase Kit (Fermentase, Hanover, Germany) according to the manufacturer’s instructions. RT-qPCR was carried out with the SYBR Green I Master Mix kit (Ampliqon, Copenhagen, Denmark) using the ABI 7500 System (Applied Biosciences, Foster City, CA). The copies of target genes were normalized to reference gene GAPDH RNA level as the internal normalization control. Relative expression was calculated using the 2-ΔΔct method. Sequences of Primers are provided in Supplementary Table 1.
Population doubling time (PDT) assay
5 × 104 MSCs transfected with SIRT1-antagoNAT at passages 2 and 7 and their relative controls were cultured in DMEM medium supplemented with 10% FBS and underwent serial passages. At the end of each passage, the cells were counted using Neubauer counting chamber. The population doubling time (PDT) was calculated using the formula: PDT=ln2*T/ln (NT/N0), in which T, NT, and N0 stand for culture time, cell number at the end of a passage and cell number at the beginning of a passage, respectively.
Cell cycle analysis
P2 and P7 MSCs transfected with SIRT1-antagoNAT and their relative controls underwent flow cytometric cell cycle analysis, 48 hours post-transfection. After fixation in 70% ethanol, washing in PBS and incubation with RNase A (20 ng/mL;Sigma-Aldrich, Steinheim, Germany) for 30 min at 37°C, the cells were stained with propidium iodide (1µg/ml; Sigma-Aldrich, Steinheim, Germany) for 5–10 min. The DNA content was measured as fluorescence intensity detected by fluorescent-activated cell sorter (FACS) (BD FACSCalibur™, BD Biosciences, San Jose, CA, USA) at 488 nm. Cells in different phases of the cell cycle were quantified with Flowjo software (Tree Star, Inc., Ashland, OR).
Colony formation assay
P2 and P7 MSCs transfected with SIRT1 antagoNAT and their relative control cells were seeded at densities of 100 and 500 viable cells/cm2 in 6-well plates. After 14 days of incubation at 37°C, the cells were fixed with ice-cold methanol for 10 min and stained with 0.5% Giemsa solution (Sigma-Aldrich, Steinheim, Germany). The mean number of colonies was quantified as a measure of colony formation potential.
Senescence-associated β-galactosidase staining
Senescence-associated β-galactosidase (SA-β-Gal) activity was detected using Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, Steinheim, Germany) according to manufacturer’s instructions. Cells were seeded in a 12-well plate at a density of 2×104 per well and incubated at 37°C with 5% CO2 for 2 days. For staining, the cells were washed in PBS, fixed for 5 min at room temperature in the fixation solution and incubated with the staining solution overnight at 37°C in the absence of CO2. The activity of SA-β-Gal was quantified by measuring the percentage of blue-stained cells out of total cells via microscopic observation.
Western blot analysis
Cell lysates were prepared in the RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 1mM protease inhibitor, PMSF (phenylmethylsulfonyl fluoride; Roche, Basel, Switzerland). BCA protein assay kit (Beyotime, Beijing, China) was used to quantify protein contents. Equal amounts of proteins were separated by SDS-PAGE and then immunoblotted onto nitrocellulose membranes. After blocking with 5% skimmed milk in TBS/T (Tris-buffered saline, 0.5% Tween 20), the membranes were incubated with primary antibodies overnight at 4°C against SIRT1, P53, GAPDH (1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA), and P16 (1:5000, Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase conjugated anti-mouse (1:5000, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-rabbit (1:5000, Abcam, Cambridge, MA, USA) secondary antibodies for 1 hour at 37°C. Detection of protein bands was performed using the Electro-Chemi-Luminescence (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ). The relative expression of target proteins were normalized to GAPDH as a reference control.
Differentiation assays
MSCs underwent osteogenic and adipogenic differentiation as described before25. For osteogenesis, MSCs were incubated in low- glucose DMEM supplemented with 10% FBS, 0.1 µM dexamethasone (Sigma-Aldrich, Steinheim, Germany), 10 µM β-glycero-phosphate (Merck, Darmstadt, Hessen, Germany), and 50 µM ascorbate (Sigma-Aldrich, Steinheim, Germany) over a period of 3 weeks, The medium was refreshed twice per week. Osteogenesis was confirmed by detection of extracellular calcium via Alizarin Red S (Sigma-Aldrich, Steinheim, Germany) staining. The cells were fixed and stained with Alizarin Red for 20 min at room temperature. Quantification of osteogenic markers including alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) were performed by qPCR. Quantification of the Alizarin Red S staining was determined using ImageJ particle analysis toolbox.
For adipogenesis, cells were incubated in high-glucose DMEM supplemented with 10% FBS, 250 nM dexamethasone, 0.5 µM 3-isobutyl‐1‐methylxanthine, 10 µg/ml insulin, and 60 µM indomethacin (all from Sigma-Aldrich) over a 3-week period. Medium refreshment was performed twice a week. Identification of adipocytes was performed by Oil red O staining. The cells were fixed and stained with 0.5% Oil Red O (Sigma-Aldrich, Steinheim, Germany) in methanol for 20 min at room temperature. Markers of adipogenic lineage including peroxisome proliferator activated receptor α (PPARα), PPARγ, and PPARγ coactivator 1 alpha (PGC1α) were quantified by qPCR. Quantification of the Oil Red O staining was determined using ImageJ particle analysis toolbox.
Alkaline Phosphatase Quantification
For quantification of ALP activity, ALP-LiquiColor assay kit (Merck Millipore, Darmstadt, Germany) was used and the absorbance of released p-nitrophenyl phosphate as the substrate was measured at 405 nm in a microplate reader. Total protein content was determined using BCA protein assay kit and used as a reference for normalization to calculate relative alkaline phosphatase activity.
Calcium content assay
Cells were homogenized in 0.5 N HCl (Merck, Darmstadt, Germany) for an hour at 25°C and the extracts were cleared by centrifugation. Calcium Liquicolor kit (Parsazmun, Tehran, Iran) was used to quantify the calcium content of the supernatants, according to the manufacturer’s instructions. The absorbance was read at 570 nm and the total calcium concentration was measured by comparison with serial dilutions of the standard solution.
Statistical analysis
Statistical analysis was performed using Graphpad Prism 7.03 software (San Diego, Calif). All of the results were expressed as mean ± SD. Differences between two groups were tested by two-tailed Student’s t test. The Pearson correlation coefficient was used to analyze the strength of the correlation between the gene expression levels and passage number. P values less than 0.05 were considered as statistically significant.