Effect of oxidative stress and 3‐hydroxytyrosol on DNA methylation levels of miR‐9 promoters

Osteoarthritis (OA) is the most common form of arthritis with increasing prevalence. Although it is a multifactorial disease, it is accepted that ageing can induce the onset of OA and has been proposed as the main risk factor of this pathology.1 The main reactive oxygen species (ROS) detected in chondrocytes are peroxynitrite (ONOO−) and hydrogen peroxide (H2O2), and when their overproduction is not counter‐balanced by an effi‐ cient antioxidant system, the oxidative stress condition occurs that enhances cartilage degeneration and OA.2 Furthermore, H2O2 supplementation has been shown to elicit oxidative stress in chondrocytes.3,4 So far, innovative strategies of treatments with no side effects need to be elucidated. For this purpose, diet‐ derived natural compounds raised a noteworthy interest due to their preventive and therapeutic action in OA.5,6 Hydroxytyrosol (HT), a polyphenol contained in olive oil and derivatives, has been proposed as a fascinating molecule able to reduce oxida‐ tive stress‐induced cellular damage and to change epigenetic signature by modulating a microRNA (miR) in chondrocytes.7,8 According to our findings, miR‐9 results to be overexpressed under chondrocyte exposure to H2O2 and miR‐9 dysregulation under TGF‐β1‐dependent ROS increase has been reported in other cell models,9,10 thus confirming its susceptibility to redox state and oxidative stress. However, the priming mechanism by which oxidative stress and HT could trigger these modulations is still lacking. Indeed, the molecular key underlying regulation of miR expression in OA is not completely clear and needs further investigation. In humans, miR‐9 is transcribed from three inde‐ pendent genomic loci mapping to chromosomes 1q22 (MIR9‐1), 5q14.3 (MIR9‐2) and 15q26.1 (MIR9‐3). Our present work sought to clarify this aspect by studying DNA methylation of the three miR‐9 promoters in response to H2O2 and HT treatments in C‐28/ I2 chondrocytes.


| INTRODUC TI ON
Osteoarthritis (OA) is the most common form of arthritis with increasing prevalence. Although it is a multifactorial disease, it is accepted that ageing can induce the onset of OA and has been proposed as the main risk factor of this pathology. 1 The main reactive oxygen species (ROS) detected in chondrocytes are peroxynitrite (ONOO − ) and hydrogen peroxide (H 2 O 2 ), and when their overproduction is not counter-balanced by an efficient antioxidant system, the oxidative stress condition occurs that enhances cartilage degeneration and OA. 2 Furthermore, H 2 O 2 supplementation has been shown to elicit oxidative stress in chondrocytes. 3,4 So far, innovative strategies of treatments with no side effects need to be elucidated. For this purpose, dietderived natural compounds raised a noteworthy interest due to their preventive and therapeutic action in OA. 5,6 Hydroxytyrosol (HT), a polyphenol contained in olive oil and derivatives, has been proposed as a fascinating molecule able to reduce oxidative stress-induced cellular damage and to change epigenetic signature by modulating a microRNA (miR) in chondrocytes. 7,8 According to our findings, miR-9 results to be overexpressed under chondrocyte exposure to H 2 O 2 and miR-9 dysregulation under TGF-β1-dependent ROS increase has been reported in other cell models, 9,10 thus confirming its susceptibility to redox state and oxidative stress. However, the priming mechanism by which oxidative stress and HT could trigger these modulations is still lacking. Indeed, the molecular key underlying regulation of miR expression in OA is not completely clear and needs further investigation. In humans, miR-9 is transcribed from three independent genomic loci mapping to chromosomes 1q22 (MIR9-1), 5q14.3 (MIR9-2) and 15q26.1 (MIR9-3). Our present work sought to clarify this aspect by studying DNA methylation of the three miR-9 promoters in response to H 2 O 2 and HT treatments in C-28/ I2 chondrocytes.

| cDNA synthesis and Real-Time PCR
RNA pellets were treated with DNAse (DNA-free, Ambion) and quantified by using RiboGreen RNA quantitation reagent (Molecular Probes). MicroRNA reverse transcription was conducted with TaqMan MicroRNA RT kit (Life Technologies), and qPCR was performed with TaqMan Universal Mastermix (Life Technologies) following kit instructions. Mature miR quantification was performed by using TaqMan MicroRNA Assays for miR-9 and U6 snRNA (internal control), according to manufacturer's recommended protocols.
After washes, membranes were incubated with horseradish peroxidase-conjugated anti-mouse (Santa Cruz Biotechnology) IgG for 1 hour. The chemiluminescent signals were detected using an ECL system (Luminata ™ Crescendo, Millipore).

| Statistical analysis
Data are reported as mean ± standard deviation (SD). Means were compared with GraphPad Prism5 statistical software (GraphPad Software, Inc). Differences were considered statistically significant at P < .05.

| MiR-9 expression is increased by H 2 O 2induced promoter demethylation
Our previous study 7 showed that miR-9 levels increase after treatment with H 2 O 2 and decrease with HT. In order to evaluate if miR-9 expression could be influenced by methylation status of its promoters in our cellular model (as drawn in Figure 1A), 5′-Aza, a DNA methyltransferase (DNMT) inhibitor, was used. The levels of miR-9 increased after 5′-Aza treatment in a dose-dependent manner ( Figure 1B). Therefore, the status of CpG islands surrounding promoters of miR-9 genes is important for the regulation of gene expression.
Promoter methylation levels of miR-9-1, miR-9-2 and miR-9-3 were assessed in response to HT and/or H 2 O 2 by using methylation-specific PCR (MSP). As shown in Figure 1C

| SIRT1 silencing determines demethylation of miR-9 promoters
SIRT1 has been reported as a genuine target of miR-9 and SIRT1 levels decreased in response to H 2 O 2 -induced oxidative stress. 7 To determine whether SIRT1 could modulate methylation of miR-9 promoters in a negative feedback loop, C-28/I2 cells were depleted of SIRT1 by RNA interference. Protein samples were immunoblotted with SIRT1 antibody to test the transfection outcome ( Figure 2A). Then, sample DNA was extracted and analysed by MSP. As shown in Figure 2B, SIRT1 knockdown changes methylation status of promoters by hypomethylating all three of them. However, we did not observe a corresponding increase in miR-9 expression in SIRT1-silenced cells ( Figure 2C)

| D ISCUSS I ON
In our previous work, we demonstrated that HT, a polyphenol found in olives and derivatives, can prevent oxidative stress-induced cell death and autophagy dysfunction by modulating miR-9 availability and its cognate target SIRT1. Thus, miR-9 has been identified as a crucial factor orchestrating the molecular response to H 2 O 2 and HT in chondrocytes. 7,13 Dysregulated levels of miR-9 in OA patients have been published, 15 and besides SIRT-1, other targets associated with OA pathogenesis have been reported, including MMP-13 16 and monocyte chemo-attractant protein 1-induced protein 1 (MCPIP-1). 17 Nevertheless, the fuse triggering the variations of miR expression was unknown. A genome-wide DNA methylation study performed in OA cartilage identified miR-9 as an OA susceptibility gene among other factors. 18 To explore whether our treatments could influence miR-9 expression by modifying methylation status of CpG islands surrounding the three promoters of miR-9 genes, we treated the cells with the DNMT inhibitor 5′-Aza and detected a dose-dependent increase in miR-9 levels. Furthermore, all three miR-9 promoters were shown to be hypomethylated in cells treated with H 2 O 2 and hypermethylated in cells treated with HT alone or both. Taken together, these results suggest that these treatments modulate miR-9 expression by exerting opposite effects on the promoter methylation status, with oxidative stress reducing and HT rescuing and sustaining the hypermethylation of CpG islands. Since no methylation differences among the three promoters have been highlighted, we could speculate that all the three genes contribute to the expression levels of miR-9.
Since miR-9 reduces its direct target SIRT1, as demonstrated by luciferase assay, 7 we investigated whether, in turn, SIRT1 could be implicated in the modulation of miR-9 levels in a negative feedback loop. However, miR-9 promoter hypomethylation induced by SIRT1 F I G U R E 2 MiR-9 promoters are demethylated by SIRT1 silencing without influencing gene expression. A, Western blotting analysis of SIRT1 and β-ACTIN. Representative images and relative quantifications are shown (n = 4 independent experiments). B, MSP analysis for methylated and unmethylated sequences of miR9-1, miR9-2 and miR9-3. C, qRT-PCR analysis of miR-9 levels in SIRT1-silenced cells (n = 4 independent experiments). Values are expressed as mean ± SD, ***P < .001 silencing through RNA interference did not correspond to an increase in miR-9 expression. Thus, demethylation of miR-9 promoters can favour but per se may not be sufficient to promote miR-9 expression.
It may be hypothesized that miR-9 expression requires the involvement of some transcription factors, triggered upon oxidative stress or 5'-aza-induced general hypomethylation, but not following just SIRT1 silencing that may elicit hypomethylation restricted to miR-9 promoters. If previous work 7 elucidated the role of this miR in the H 2 O 2 -promoted cell death and in the protective effect of HT in chondrocytes, these new findings provide the upstream mechanism influencing the variations of miR-9 expression. The identification of a miR able to address the cell fate in response to a protective and/or stress agent opens novel perspectives in the field of molecular therapy for degenerative diseases, such as OA. Indeed, a better understanding of the interaction of different epigenetic levels in OA pathogenesis, including promoter methylation status, miR expression and transcriptome changes, could be useful to prime further investigations for a miR-based strategy with nutraceutical support in the treatment of this disease.

ACK N OWLED G EM ENTS
This work was supported by grants from University of Bologna (RFO) and Ministero della Salute, Italy (Fondi Cinque per Mille, year 2016).

CO N FLI C T O F I NTE R E S T
The authors have declared that there is no conflict of interest.

AUTH O R CO NTR I B UTI O N
SD designed the experiments. SD and SC performed the experiments. SD, SC, RMB and FF analysed and interpreted the data. SD, SC, RMB and FF contributed in writing and approving the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.