If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, ItalyDipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Bologna, Italy
Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Bologna, ItalyLaboratorio di Immunoreumatologia e Rigenerazione Tissutale, Istituto Ortopedico Rizzoli, Bologna, Italy
Address correspondence and reprint requests to: F. Flamigni, Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Via Irnerio 48, 40126, Bologna, Italy.
Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1).
Methods
Human primary and C-28/I2 chondrocytes were pre-treated with 100 μM HT 30 min before 100 μM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes.
Results
MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2.
Conclusions
These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.
are only based on palliative treatments that focus on symptoms, e.g., pain and inflammation, and mainly consist of analgesics and non-steroidal anti-inflammatory drugs (NSAIDs)
. Unfortunately they lack efficacy in slowing disease progression and, also, result in several side effects, mostly gastrointestinal and cardiovascular injuries.
MicroRNAs (miRs) are an abundant, evolutionary conserved subfamily of short non-coding RNAs (22–25 nt) that are identified as potent post-transcriptional regulators
. Recently the great potential of miRs as important regulators of a specific target and/or entire cellular processes has received much attention and also in the field of OA research many investigators are striving to identify the fine crosstalk between deregulated pathways and miRs, thereby discovering new intriguing molecular therapeutical targets
. Studies on miR deregulation in OA are performed by comparing the expression of these molecules between OA tissue specimens and their normal articular counterpart
. Then functional studies allow to shed light on the specific role of a deregulated miR in OA through assessment of the modulation of miR levels and subsequent evaluation of pathological phenotypes in in vitro cellular models. In particular miR-140, presently the best characterized miR implicated in OA, is reduced in OA tissue
. Moreover, SOX9, the major transcription factor implicated in the differentiation of chondrocyte phenotype and prevention of cellular hypertrophy, can regulate miR-140 levels
. Also miR-155, upregulated in OA, can contribute to the autophagy defects in OA by suppression of gene and protein expression of key autophagic regulators
On the other hand several reports suggest that nutraceuticals exert a protective role for degenerative pathologies, including cardiovascular disease, cancer, and OA
. Some nutraceuticals have shown to exhibit a role not merely as anti-oxidants and ROS scavengers, but also as efficient modulators of gene expression of key factors underlying the OA onset
. In this regard an interesting candidate molecule is hydroxytyrosol (HT), a phenolic compound endowed with a powerful anti-oxidant action, mainly found in the fruits of olive tree (Olea europaea L.) and derivatives, such as olive oil
Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction.
Hydroxytyrosol reduces intracellular reactive oxygen species levels in vascular endothelial cells by upregulating catalase expression through the AMPK-FOXO3a pathway.
. Moreover, we demonstrated that HT mediates the effect of chondroprotection by promoting the autophagy process and cytoplasm-nucleus translocation of sirtuin-1 (SIRT-1), a “longevity factor” and autophagy regulator
However to date, few nutraceuticals have been reported as modulators of specific miRs and no one in OA physiopathology. In the present work primary OA and C-28/I2 chondrocytes were employed to address the question whether HT could exert its protective effect against oxidative stress by modulating specific miRs. We show that HT protects from cell death, reduces the expression of some OA markers and rescues protein levels of SIRT-1 by impairing the up-regulation of miR-9 by H2O2.
, kindly provided by Dr. Mary Goldring, are a human cell line representative of primary chondrocytes that has been used extensively as a reproducible ‘‘in vitro’’ model to study chondrocyte physiopathology in experiments requiring large numbers of cells. With local Ethics Committee approval, primary cultures of chondrocytes were used and prepared from fragments of articular cartilage obtained from adult OA patients undergoing knee arthroplasty. The number of different primary cultures used for each experiment is detailed in Supplementary Table I. The use of the human cells in this study was in accordance with the Helsinki Declaration of 1975. Both C-28/I2 and primary chondrocytes were grown in DMEM medium supplemented with 10% fetal bovine serum as previously detailed
The cells were incubated in the absence or presence of 100 μM H2O2 for 4 or 24 h as indicated in the various figures; 100 μM HT (from Cayman Chemical) was added 30 min before H2O2, on the basis of previously published studies
. Control cells received the corresponding volume of vehicle. Viable cells were directly counted following the trypan blue exclusion test. Dead cells including the dye were reported as a percentage of the total number of cells. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) as previously mentioned
. Since the sequence DEVD represents a substrate for caspase-3 and other effector caspases, this activity has been referred to as caspase 3-like activity. Caspase activity was expressed per mg protein, and normalized to untreated controls.
Cell transfection
Primary chondrocytes and C-28/I2 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and in 96-well plates at a density of 1,28 × 103 cells/well without antibiotics. The next day, Ambion® Anti-miR™ miRNA Inhibitors, negative control #1 (antimiR-NC) (50 nM) and antimiR-9 (50 nM), and Ambion® Pre-miR™ miRNA Precursors, negative control #1 (premiR-NC) (50 nM) and premiR-9 (Life Technologies) (50 nM) were transfected into cells by using Lipofectamine RNAiMAX (Invitrogen) for 24 h. Anti-miR miRNA Inhibitors are single stranded nucleic acids designed to specifically bind to and inhibit endogenous miR molecules. Pre-miRNA precursor molecules are small, chemically modified double stranded RNA molecules designed to mimic endogenous mature miRs.
C-28/I2 and primary cells were co-transfected with either antimiR-NC or antimiR-9 and either ON-TARGETplus Human Sirt1 siRNA (25 nM) or ON-TARGETplus non-targeting pool (25 nM) (Dharmacon) by Lipofectamine® 3000 Reagent in Opti-MEM® Medium (Life Technologies) according to manufacturer's instructions. The cells were incubated for 24 h before evaluating cell viability and caspase 3-like activity.
Western blotting
In western blotting procedure equal amounts of cell extract were subjected to electrophoresis in 10% gels, blotted onto nitrocellulose membranes essentially as previously described
, and probed with primary antibody at 4°C overnight. The following antibodies were used: anti-β-actin (A5316, Sigma–Aldrich), anti-SIRT-1 (sc-74465, Santa Cruz Biotechnology). After washes, membranes were incubated with horseradish peroxidase-conjugated anti-mouse (Santa Cruz Biotechnology) IgG for 1 h. The chemiluminescent signals were detected using an ECL system (LuminataTM Crescendo, Millipore). β-actin was used as loading control. A representative image of visualized immunoreactive bands and densitometric analysis show the relative intensity of protein expression.
Bioinformatics prediction of miR candidates targeting SIRT-1
TargetScan and miRWalk databases were used to predict miR candidates targeting SIRT-1 by detecting the complementarity between their seed sequences and specific sequences in SIRT-1 mRNA.
RNA isolation, cDNA synthesis and real-time PCR
Total cellular RNAs were extracted with 700 μl TRIZOL (Invitrogen), according to manufacturer's instructions. The RNA pellets were treated with DNAse (DNA-free, Ambion, Austin, TX) and, after buffer exchange, were quantified by using RiboGreen RNA quantitation reagent (Molecular Probes, Eugene OR). The same quantity of total RNA (100 ng) was reverse transcribed by using random primers and the reagents provided with the Superscript VILO System for RT-PCR (Invitrogen). The cDNA mixture (2 μl) was used in Real time PCR analysis in a LightCycler Instrument (Roche Molecular Biochemicals) by means of the QuantiTect SYBR Green PCR kit (TaKaRa, Japan) with the following protocol: initial activation of HotStart Taq DNA polymerase at 95°C for 10 s, followed by amplification (40 cycles: 95°C for 5 s followed by appropriate annealing temperature for each target as detailed below kept for 20 s). The protocol was concluded by melting curve analysis to check amplicon specificity. The following primers (from Invitrogen) were used: GAPDH (NM_002046) 579–598F and 701–683R; MMP-13 (NM_002427) 496–511F and 772–756R; RUNX-2 variant transcript 3 (NM_004348) 864–883F and 968–949R, RUNX-2 variant transcripts 2 (NM_001015051) and 1 (NM_001024630) 716–735F and 820–801R; VEGF variant transcripts7 (NM_001033756.1), 6 (NM_001025370.1), 5 (NM_001025369.1), 4 (NM_001025368.1), 3 (NM_001025367.1), 2 (NM_003376.4), and 1 (NM_001025366.1), 1144–1126 (forward) and 1063–1079 (reverse). Primers were annealed at 56°C. The amount of mRNA was normalized to GAPDH expression in each sample and referred to untreated, control sample.
MicroRNA reverse transcription was conducted with TaqMan MicroRNA RT kit (Life Technologies) and qPCR was performed with TaqMan Universal Master mix (Life Technologies) following kit instructions. Mature miR quantification was performed using TaqMan MicroRNA Assays for miR-9 and U6 snRNA (internal control), according to manufacturer recommended protocols (Applied Biosystems). 10 ng of total RNA, 50 nM stem-loop RT primer, RT buffer, 0.25 mM each dNTP, 3.33 units/ml MultiScribe reverse transcriptase (RT), and 0.25 units/ml RNase inhibitor were used in 15-μL RT reactions for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C, using the TaqMan MicroRNA reverse transcription kit (Applied Biosystems). For real-time PCR, 1.33 μl (1:15 dilution) of cDNA, 0.2 mM TaqMan probe, 1.5 mM forward primer, 0.7 mM reverse primer, and TaqMan Universal PCR Master Mix (Applied Biosystems) were added in 20 μl reactions for 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C.
Luciferase reporter assay
To confirm that the regulation of SIRT-1 expression is mediated by the matching between its 3′-UTR and miR-9, we used pEZX-MT06 reporter vector that contains miR binding sequences of SIRT-1 3′UTR (GeneCopoeia, Rockville, MD) in combination with Luc-Pair™ Duo-Luciferase HS Assay Kit (GeneCopoeia, Rockville, MD). C-28/I2 cells were co-transfected with either plasmid carrying wild type 3′UTR of SIRT-1 (3′UTR wt), or 3′UTR without the seed-complementary sequence (identified by TargetScan) (3′UTR mut1), or 3′UTR without the seed-complementary sequence and the 3′ pairing sequence (identified by TargetScan) (3′UTR mut2), and either premiR-9 or premiR-NC. The transfection was carried out in antibiotic-free medium using Lipofectamine 3000 according to the manufacturer's protocol (Invitrogen). The dual luciferase activity of transfected cells were measured by using WallacVictor2 1420. The results were expressed as percentage of normalized firefly luciferase activity against Renilla luciferase activity. The results were expressed as percentage reduction of luciferase activity in premiR-9 transfected cells in relation to 100% activity for cells transfected with premiR-NC.
Statistical analysis
All data shown were achieved by carrying out the indicated number of independent experiments for C-28/I2 cells or the indicated number of patients for primary chondrocytes (Supplementary Table I). Each harvested experimental data is the mean of a technical triplicate. To evaluate normal distribution of the data, we performed the Kolmogorov–Smirnov test and homogeneity of variance using Bartlett's test. Error bars shown in the graphs are expressed as means ± 95% confidence interval. Comparisons between groups were performed with Student's t test. GraphPad Prism 5 statistical software (GraphPad Software, Inc, San Diego, CA) was used for all statistical analyses. Since no previous findings were available no power calculations were performed.
Results
Opposite variations of miR-9 and Sirt-1 levels in response to H2O2 and HT treatments in human primary chondrocytes and C-28/I2 cells
In our previous works, HT was found to be able to reduce H2O2-induced cell death and DNA damage in human primary chondrocytes and C-28/I2 cells
. The effects of HT and H2O2 treatments occurred at least in part through the mediation and regulation of SIRT-1, even though no notable changes in SIRT-1 mRNA levels were observed
. In the present study the expression of SIRT-1 protein was evaluated by immunoblotting [Fig. 1(A)]. Interestingly H2O2 treatment reduced, whereas HT alone or together with H2O2 increased the expression of SIRT-1 protein in both cell cultures. This intriguing result spurred us to investigate the molecular mechanism underlying the post-transcriptional regulation of SIRT-1 gene expression resulting in the changes of SIRT-1 protein levels by HT and H2O2. In this regard, miRs represent an intensively studied class of short non-coding RNAs that is able to regulate gene expression post-transcriptionally. By exploiting miR-target prediction tools of different databases available online (TargetScan, miRanda, miRWalk), some miRs, including miR-22, miR-34 and miR-9, were selected because of the presence of seed-complementary sequences in SIRT-1 3′UTR and on the basis of the probability of prediction and validation. We evaluated miR levels by using specific primers (TaqMan microRNA assays) able to detect mature miR sequences discriminating them from their precursors (primiR, premiR). No changes in miR-22 and miR-34 amounts were observed after treatments, but significant variations were appreciated in miR-9 levels: H2O2 led to a substantial increase of miR-9 after 4 h of treatment and HT reduced this effect in both primary chondrocytes and C-28/I2 cells [Fig. 1(B)]. The site of matching identified by TargetScan browser was shown in the panel C of Fig. 1. The opposite variations of miR-9 and SIRT-1 indicate that the latter is a possible target of miR-9 as predicted by the site of matching.
Fig. 1Opposite variations of SIRT-1 and miR-9 levels in response to H2O2 and HT treatments in human primary and C-28/I2 chondrocytes. Cell cultures were pre-incubated in the absence or in the presence of 100 μM HT for 30 min before addition of 100 μM H2O2. After 24 h of incubation cells were harvested for SIRT1 detection by western blotting. Representative images and relative quantification for SIRT1/β-actin ratio are shown (A). After 4 h of incubation samples were collected for miR-9 quantification by qPCR analysis (B). Site of matching between SIRT1 3′UTR and miR-9 sequences (provided by TargetScan database) is shown (C). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
Impact of miR-9 silencing on H2O2-induced cell death in human primary chondrocytes
Next, we evaluated the role of miR-9 in cell death elicited by H2O2 as well as in the protection afforded by HT in human primary chondrocytes. First, we tested the silencing efficiency of Ambion® Anti-miR™ miRNA Inhibitors by using qPCR and showed that antimiR-9 significantly reduced miR-9 levels in H2O2 -treated and -untreated cells [Fig. 2(A)]. We found out that miR-9 silencing rescued protein levels of SIRT-1 reduced by H2O2 treatment [Fig. 2(B)]. Accordingly, antimiR-9 transfection markedly reduced cell death and completely prevented the increase of caspase 3-like activity induced in antimiR-NC transfected cells by H2O2 [Fig. 2(C)]. Then, in order to estimate the importance of SIRT-1 in the protection obtained by reducing miR-9 levels, primary chondrocytes were silenced for both miR-9 and SIRT-1 and treated with H2O2. SIRT-1 silencing was ascertained by western blot analysis of SIRT-1 protein (Supplementary Fig. 1). Figure 2(D) shows that SIRT-1 knockdown decreased the protection afforded by antimiR-9 transfection vs H2O2-induced cell death. Indeed, we found a significant difference of percent of dead cells and caspase activity between SIRT-1 plus miR-9 -silenced cells and miR-9 only-silenced cells when treated with H2O2. Although antimiR-9 transfection was still able to reduce H2O2-induced cell death in SIRT-1-silenced cells, a partial loss of protection was observed when compared with cells expressing SIRT-1. Therefore it is conceivable that miR-9 may influence more targets, in addition to SIRT-1, implicated in the control of apoptotic cell death mediated by oxidative stress.
Fig. 2Impact of the miR-9 silencing on the toxicity of H2O2 in human primary chondrocytes. Primary cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM). After 4 h incubation cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without H2O2, cells were collected for SIRT1 detection by western blotting. Representative images and relative quantification of SIRT1/β-actin ratio are shown (B). Alternatively, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (C). In addition, cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and siCTRL or siSIRT-1 (25 nM). After 24 h incubation with or without 100 μM H2O2, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (D). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
Impact of miR-9 overexpression on H2O2-induced cell death and HT-mediated cytoprotection in human primary chondrocytes
Then, we verified the efficacy of Ambion® Pre-miR™ miRNA Precursors transfection by performing qPCR: as shown in Fig. 3(A), these oligonucleotides were able to raise dramatically mature miR-9 levels. We observed that simultaneously pre-miR-9 transfection decreased protein expression of SIRT-1 in HT-treated and -untreated cells [Fig. 3(B)]. Moreover, premiR-9 enhanced cell death compared to correspondent control cells and HT only partially preserved its protective effect against cell death induced by oxidative stress [Fig. 3(C)]. Consistently with the changes in cell death rate, Fig. 3(C) shows that premiR-9 transfection enhanced caspase 3-like activity and led to a partial loss of caspase inhibition provoked by HT pre-treatment.
Fig. 3Impact of premiR-9 transfection on the HT-mediated protection in human primary chondrocytes. Cells were transfected for 24 h with premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without HT, cells were collected for SIRT-1 detection by western blotting. Representative images and relative quantification for SIRT-1/β-actin ratio are shown (B). Alternatively after 24 h incubation with 100 μM H2O2 or 100 μM HT, cells were counted to assess cell viability by trypan blue exclusion test or harvested and analyzed for caspase activity (C). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
Impact of miR-9 silencing and overexpression on H2O2-induced cell death and HT-mediated protection in C-28/I2 cells
In order to confirm these observations in the C-28/I2 cell line, transfections with Ambion® Anti-miR™ miRNA Inhibitors and Pre-miR™ miRNA Precursors were also performed in these cells and their efficiency validated by qPCR, as shown in Fig. 4(A). Then, we evaluated the protein levels of SIRT-1 in transfected cells and found that miR-9 silencing significantly increased the protein amount compared to H2O2-treated cells [Fig. 4(B)]. It may be noted that, as expected, no notable change was observed in control cells, likely due to the lower basal levels of miR-9 in C-28/I2 cells compared to human primary chondrocytes. Conversely, premiR-9 transfection dramatically decreased SIRT-1 protein expression with or without HT addition [Fig. 4(B)]. Furthermore, miR-9 knockdown led to a complete rescue of cell viability after H2O2 treatment. PremiR-9 transfection elicited cell death in control, HT and H2O2-treated cells and abolished HT protection from cell death mediated by H2O2 [Fig. 4(C)]. Co-transfection experiments performed in order to silence SIRT-1 and/or miR-9 confirmed that SIRT-1 contributes in antimiR-9 protection from cell death [Fig. 4(C)], as SIRT-1 knockdown significantly increased cell death in antimiR-9 co-transfected compared with antimiR-9 only transfected cells. The efficacy of silencing SIRT-1 in C-28/I2 cells was documented in our previous report
Fig. 4Impact of antimiR-9 and premiR-9 transfection on H2O2-induced cell death and HT-mediated protection in C-28/I2 cells. Cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and miR-9 levels were determined by qPCR (A). After 24 h incubation with or without HT, cells were collected for SIRT1 detection by western blotting. Representative images and relative quantification for SIRT1/β-actin ratio are shown (B). Alternatively after 24 h incubation with 100 μM H2O2 or 100 μM HT, cells were counted to assess cell viability by trypan blue exclusion test (C). In addition, cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and siCTRL or siSIRT1 (25 nM). After 24 h incubation with or without 100 μM H2O2, cells were counted to assess cell viability by trypan blue exclusion test (D). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
To demonstrate the direct link between miR-9 and SIRT-1 as its actual target, we performed a luciferase-based gene reporter assay. We transfected three different gene sequences [Fig. 5(A)] inserted in the plasmid provided by Genecopoeia: 3′UTR mut1 consists of 3′UTR SIRT-1 without miR-9 seed sequence, as identified by TargetScan bioinformatic tool; 3′UTR mut2 lacks seed sequence and the miR-9 3'pairing sequence; 3′UTR wt (wild type) corresponds to the full 3′UTR sequence of SIRT-1. 24 h after transfection, the enzymatic activity of reporter protein was evaluated and only cells transfected with the full 3′UTR showed a decreased signal (around 50%) of bioluminescence [Fig. 5(B)]. Overall these data confirm that SIRT-1 is a genuine target of miR-9.
Fig. 5SIRT1 is a direct target of miR-9 in C-28/I2 cells. Three different sequences were designed and provided within pEZX-MT06 reporter vector (Genecopoeia); a first sequence deleted of seed sequences (3′UTR mut1); a second one deleted of miR-9 3′ pairing sequence as well as of seed sequence (3′UTR mut2); a last one with the full 3′UTR sequence (3′UTR wt) (A). Cells were co-transfected with either plasmid carrying 3′UTR mut1, 3′UTR mut2 or 3′UTR wt, with premiR-9 or premiR-NC. The dual luciferase activity of the transfected cells was detected and the luciferase activity was normalized on renilla activity (B). Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
MiR-9 levels influence the expression of OA-related genes in primary OA chondrocytes
The effect of variations in miR-9 levels on the expression of selected genes known to be involved in OA pathogenesis and already reported as responsive to H2O2 and HT treatments was investigated by qRT-PCR. Figure 6 shows that miR-9 knockdown significantly decreased mRNA levels of MMP13, VEGF and RUNX2 in both non-treated and H2O2-treated cells for 4 h. Accordingly premiR-9 transfection significantly increased the expression of MMP13, VEGF and RUNX2 genes compared to premiR-NC in HT-treated and -untreated cells (Fig. 6), even if the increase was less marked in chondrocytes treated with HT.
Fig. 6MiR-9 levels influence the expression of OA-related genes in primary OA chondrocytes. Cells were transfected for 24 h with antimiR-9 or antimiR-NC (50 nM) and premiR-9 or premiR-NC (50 nM). After 4 h incubation with 100 μM H2O2 or 100 μM HT cells were harvested and analyzed by qRT-PCR for the amount of MMP-13 mRNA, VEGF mRNA and RUNX-2 mRNA. Values are expressed as 95% confidence intervals, and P values of the differences are shown above comparisons between groups.
Previous research from our group demonstrated that HT, a nutraceutical compound found in olives and derivatives, contrasts OA related-changes and protects against oxidative stress in human primary and C-28/I2 chondrocytes
. In order to further characterize the molecular mechanisms underlying the protective effect of HT against H2O2-induced cell death, we aimed at identifying a common factor targeted by both HT and H2O2 and able to address chondrocyte fate. As we had previously showed that SIRT-1 was involved in HT protection of chondrocytes from H2O2-induced cell death, we speculated that miR-9 was the missing piece of puzzle. Indeed the seed-complementary sequence to miR-9 in SIRT-1 3′UTR (5′ UCUUUGGU-3′) identified by TargetScan algorithm is highly conserved among species and let us hypothesize that SIRT-1 could be a genuine target of miR-9 in our experimental model. Indeed, luciferase reporter assay confirmed that miR-9 targets SIRT-1 in C/28-I2 cell line [Fig. 5(B)].
Consistent with our finding, Saunders et al. reported that five different miRs suppressed SIRT-1 protein expression and miR-9 was among these regulators
; however our study is the first to demonstrate that SIRT-1 mRNA is a direct target of miR-9 in chondrocytes. Accordingly, in our cell models miR-9 and SIRT-1 levels varied in opposite ways. Besides, not only H2O2 led to substantial increase of miR-9, whereas HT reduced this effect [Fig. 1(B)], but transfection experiments with premiR-9 and antimiR-9 showed that miR-9 increase mediates cell death induced by H2O2 and conversely miR-9 down-regulation mediates the protective effect of HT, as observed in both human primary chondrocytes and C/28-I2 cell line.
Moreover, the silencing of SIRT-1 in antimiR-9-transfected chondrocytes still resulted in some additional loss of protection of H2O2-treated chondrocytes, even though less marked [Fig. 2, Fig. 4]. Thus it may be concluded that SIRT-1 is only one determinant underlying chondrocyte protection and, actually, miR-9 is the real hub of cellular response to HT and H2O2 treatments. Indeed, as it is conceivable that this polyphenol acts through several mechanisms of action, including miR-9 regulation demonstrated in this paper, miR-9 too may influence several target genes including SIRT-1, which is already recognized as a key factor in the protection of chondrocytes from oxidative stress
. Overexpression of miR-9 in OA following significant epigenomic changes has also been confirmed by a recent study exploiting a Genome wide DNA Methylation analysis
Jones et al. found up-regulated levels of miR-9 and miR-98 and down-regulated levels of miR-146 in OA samples and since they found that overexpression of these miR leads to reduced IL-1β mediated TNF-α production they hypothesize that these miRs exert a protective role during late-stage OA
. Quite recently Makki et al. also confirmed an enhanced expression of miR-9 in damaged OA cartilage, but showed that miR-9 promotes IL-6 expression, which plays an important role in OA pathogenesis, by targeting MCPIP1 in IL-1β-stimulated human chondrocytes
MicroRNA-9 promotion of Interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in Interleukin-1 beta-stimulated human chondrocytes.
. Moreover in IL-1β-stimulated human chondrocytes miR-9, miR-188 and let7e levels increased, but only miR-9 proved to be a negative post-transcriptional regulator of membrane proteins that control the balance between extracellular levels of Pi and PPi, suggesting that miR-9 contributes to the mineralization process and the acquirement of an altered chondrocyte phenotype
The ROS-sensitive microRNA-9/9* controls the expression of mitochondrial tRNA-modifying enzymes and is involved in the molecular mechanism of MELAS syndrome.
confirming that miR-9 responds to perturbations in the redox state. Overall these results indicate that miR-9 acts as the main player of H2O2-induced changes in human chondrocytes and is likely involved in oxidative stress-related and possibly other molecular aspects of OA pathology.
The involvement of miRs across human diseases has been widely reported and has motivated the development of miRs-based therapies. MiRs are promising targets as a single miR is able to regulate multiple genes in dysregulated pathways in a disease, whereas classical therapies based on “one gene-one target” approach do fail. On the other hand natural agents in a healthy diet have shown pleiotropic beneficial effects against several pathways found to be deregulated in many degenerative diseases, including cancer
. Interestingly Di Francesco et al. showed that miR-23a and miR-301a expression was found to be reduced after either single or 10-day long extra virgin olive oil administration in rats
Consistently with this panorama, our study highlights the epigenetic potential of a bioactive compound, HT, able to manage gene expression of different factors implicated in OA pathogenesis. A more extensive investigation on the epigenetic mechanisms underlying the pathogenesis of chronic degenerative diseases such as OA, will allow the identification of gene expression changes in response to environmental stimuli, such as nutraceutical diet integration, with the aim to exploit them in the attempt to restore a healthy phenotype. Thus, the identification of a single miR, simultaneously involved in several disease-related pathways, discloses a potent therapeutic target. Indeed the unveiling of bioactive compounds able to influence miR network may open a new area of research with promising potential to explore.
Authors' contributions
FF had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design: SD, SC, RMB, FF. Acquisition of data: SD, SC, SG. Analysis and interpretation of data: SD, SC, RMB, FF. Manuscript preparation and approval: SD, SC, SG, RMB, FF.
Conflict of interest
None.
Grant funding
This work was supported by University of Bologna (RFO), Fondazione del Monte di Bologna e Ravenna and Ministero dell'istruzione, dell'Università e della Ricerca, Italy (FIRB grant RBAP10KCNS).
Appendix A. Supplementary data
The following are the supplementary data related to this article:
Supplementary Fig. 1SIRT-1 knockdown in human primary chondrocytes. Cells were transfected for 24 h with siCTRL or siSIRT1 (25 nM) and then harvested for SIRT-1 detection by western blotting. Representative image and relative quantification of SIRT-1/β-actin ratio are shown. Values are expressed as 95% confidence intervals, and P value of the difference is shown above comparison between groups
Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction.
Hydroxytyrosol reduces intracellular reactive oxygen species levels in vascular endothelial cells by upregulating catalase expression through the AMPK-FOXO3a pathway.
MicroRNA-9 promotion of Interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in Interleukin-1 beta-stimulated human chondrocytes.
The ROS-sensitive microRNA-9/9* controls the expression of mitochondrial tRNA-modifying enzymes and is involved in the molecular mechanism of MELAS syndrome.
30425-3&id=gr1.jpg){kind=link}
30425-3&id=gr2.jpg){kind=link}
30425-3&id=gr3.jpg){kind=link}
30425-3&id=gr4.jpg){kind=link}
30425-3&id=gr5.jpg){kind=link}
30425-3&id=gr6.jpg){kind=link}
30425-3&id=figs1.jpg){kind=link}