Association of circulating microRNAs with prevalent and incident knee osteoarthritis in women: the OFELY study

Osteoarthritis (OA) is the most frequent chronic musculoskeletal disease affecting approximately 40% of adults aged 70 years and over [1]. It is considered as a slowly progressive disease degrading all tissues of the affected joint, appearing as a destruction of the cartilage, the hallmark of OA, but also a mild-to-moderate synovial inflammation and an alteration of the subchondral bone structure. So far, the gold standard to estimate the extent of the disease has been plain radiography, but the poor sensitivity and the relatively large precision error lead to disease detection only when significant cartilage degradation has already occurred. Measuring circulating biomarkers appears as a potential non-invasive approach to diagnose the disease and prognosticate its evolution. Research has focused on the structural components of the cartilage matrix, especially type II collagen degradation markers [2]. Because of the large overlap between biomarker concentrations in controls and OA patients, the diagnosis of OA in an individual patient cannot be made by currently available biomarkers. Given the limitations of the tools that are currently available for OA assessment, there is considerable interest in the identification of specific biological markers that reflect quantitative and dynamic variations in joint tissue remodeling. For this purpose, microRNAs (miRNAs) are new potential targets. These small non-coding RNAs of 22–28 nucleotides in length can silence gene expression by binding to target messenger RNA (mRNA) repressing the translation. This regulation process is redundant because 1 miRNA can inhibit a large number of mRNA, and 1 gene can be targeted by multiple miRNAs. The miRNAs are important players in maintaining the health of all joint tissues including the cartilage, ligament, tendon, and muscle. Some of them have a potential role in regulating homeostasis of joint tissues in the context of OA [3]. Several in vitro and in vivo studies have reported the miRNA involvement in the OA onset and progression, by targeting cartilage-associated genes [4]. They regulate the expression of genes involved in pathways altered in OA chondrocytes such as apoptosis [5], expression levels of MMPs and ADAMTS [6‐8], and chondrocyte signaling [9, 10]. To date, it has been reported that about 80 miRNAs are involved in the pathology of OA and that their circulating level in patients might reflect the underlying disease state [4]. Indeed, circulating miRNAs are considered to be attractive molecules to serve as biomarkers because they are easily accessible, stable in biofluids, and quantifiable with a high degree of sensitivity and specificity. Circulating miRNAs are protected from degradation because they circulate bound to proteins such as Argonaute 2 and nucleoplasmin or inside microvesicles such as exosomes [11‐13]. Finally, although the expression of miRNAs has been well documented in OA cartilage, it remains difficult to understand to which extent circulating miRNAs reflect the tissue alteration. Moreover, the aberrant expression levels previously reported in circulating miRNAs differ considerably between clinical studies due to the different characteristics of these settings [14‐22].

In this study, a first screening phase was conducted by comparing the serum miRome in subjects with (n = 10) and without (n = 10) knee OA in the OFELY cohort by next-generation sequencing (NGS) analysis. Then, on the basis of our NGS data and on the previously reported function of miRNAs in the OA process, both in basic and/or clinical research, we have selected 19 candidate miRNAs for quantitative real-time polymerase chain reaction (RT-qPCR) analysis. By conducting the validation phase of these candidate miRNAs in the OFELY cohort, we report that miR-146a-5p and miR-186-5p are significantly associated with prevalent and incident knee OA, respectively.

In the present work, we have studied the serum miRNA profiling of women with knee OA to identify potential non-invasive biomarkers of the disease. We first screened the serum miRNAs with significant differences in the expression between women with knee OA and healthy controls by a NGS approach. Then, we have conducted a validation study in the OFELY cohort of women having an incident or prevalent knee OA with a long-time follow-up. In this, we analyzed by RT-qPCR whether the 3 differentially expressed miRNAs (139-5p, 200a-3p, 1299) in patients with knee OA in the screening step were also aberrantly expressed in the women with incident and prevalent knee OA of the validation cohort. In addition, miR-375 has been chosen because of its high expression level and a tendency to be differentially expressed in our NGS approach despite the absence of significant difference after FDR correction. Moreover, we have measured the expression levels of 15 additional miRNAs previously highlighted as having a dysregulated expression in patients with knee OA. The miRs-29 family (a, b, c) and miR-199a-3p were chosen for their activity as key regulators of chondrocyte gene expression with aberrant expression in OA cartilage [29‐31]. The miRNAs let-7e-5p, 16-5p, 93-5p, 126-3p, 132-3p, 146a-5p, 184, 186-5p, 195-5p, 345-5p, and 885-5p were selected because previous studies analyzing circulating miRNAs in the serum and plasma [14, 17, 18] have reported their dysregulated expression in patients with knee OA (see Table 4).

The NGS approach revealed that miR-139-5p, miR-1299, and miR-200a-3p had levels of expression significantly different between OA patients and controls. However, we did not validate these 3 miRNAs in the second step of the analysis when we measured their level in the largest validation cohort. This lack of validation might come from the small number of samples in the discovery step (10 OA vs 10 non-OA) even if this number is comparable to those used in previous studies (Beyer: 13 OA vs 13 non-OA [18]; Borgonio-Cuadra: 14 OA vs 5 non-OA [17]; Kong: 8 OA vs 8 non-OA [21]) and/or differences in the specificity and sensibility of the platforms used (Illumina for this study; Borgonio-Cuadra [17] and Beyer [18]: Applied Biosystems; Kong [21]: Affimetrix).

It appears that the previous studies performed with biological fluids are difficult to compare each other because of the heterogeneity of the clinical situations (see Table 2). Nevertheless, our study can be compared to that of Borgonio-Cuadra et al. [17], particularly the patient selection is based on the same criteria, radiologic KL scores of 2 and 3 and a BMI smaller than 27. However, among the ten miRNAs in common, only miR-146a-5p (miR-146) showed significant overexpression, emphasizing the challenges faced with patient heterogeneity. The reasons for this discrepancy could be the ethnic origin, Mexican vs European, enhancing the differences in exposure to environmental factors; the sex of the participants, men and women vs women only; the biological fluid used-plasma vs serum; or the mean age of participants, 55 vs 68 years.

Nevertheless, we found that miR-146a-5p expression is significantly increased in the OA group of postmenopausal women compared to healthy controls independently of age and BMI. Moreover, we reported for the first time that the risk of prevalent knee OA was significantly increased for each quartile increase in the serum level of miR-146a-5p. However, the interpretation of this increase remains challenging in regard to the dual role of miR-146a-5p in the pathophysiology of osteoarthritis. MiR-146 is expressed in chondrocytes which begin to undergo degenerative changes [32]; its expression is stimulated by pro-inflammatory cytokines in an NF-κB-dependent manner [33]. It may contribute to OA by impairing the TGF-β signaling pathway targeting Smad4 and increasing apoptosis [5, 34]. Recently, Zhang et al. showed that miR-146 facilitates OA progression by targeting Camk2d and Ppp3r2 that are required to maintain the phenotype of mature chondrocytes [33].

In contrast, miR-146 could play a protective role in OA. It is induced during hypoxia by HIF-1 and promotes autophagy by decreasing the Bcl-2 expression, an autophagy inhibitor. Autophagy has a protective function for cartilage during OA [35]. Moreover, miR-146 exerts negative control on inflammatory responses by suppressing IRAK1 and TRAF6, two molecular targets of miR-146 impairing NF-kβ activity and suppressing NF-kβ target genes such as IL-6 and TNF-α [36, 37]. Recently, Zhong et al. [38] showed that miR-146 could increase the proliferation and inhibit apoptosis of OA chondrocytes by inhibiting TRAF6 expression and suppressing the activation of the NF-κB signaling pathway. Finally, Guan et al. [39] showed that miR-146 is activated by cyclic loads in the physiological range but suppressed by mechanical overload in human articular chondrocytes. They proposed that miR-146 protects joint cartilage from degeneration through inhibition of the feedback loop of Notch-1/IL-6 during aging or trauma.

It must be noted that circulating miR-146 has been highlighted in two other studies analyzing the peripheral blood mononuclear cells. Okuhara et al. [16] found it overexpressed in patients suffering from knee OA compared to healthy controls and highly expressed in OA peripheral blood mononuclear cells from patients with early OA. Soyocak et al. [20] observed that, in patients classified according to the KL score, miR146a expression increased in the progressive stages (grades 3 and 4).

Finally, miR-146 is one of the four miRNAs with miR-223, miR-16, and miR-30b that were differently expressed in the cartilage and in the circulation with a role in the cartilage homeostasis [4]. Moreover, miR-146 is also involved in the inhibition of osteoclastogenesis [40] suggesting that its measurement in the serum could reflect the deregulations affecting the cartilage and bone tissues. Although the activity of this miRNA seems dependent on the disease phases, it appears as a key element in the OA pathophysiology, and our results suggest that miR-146a-5p could be a new biological marker for knee OA.

We report for the first time a significant association, independent of age and BMI, of serum miR-186-5p and incident knee OA risk in a population of postmenopausal women followed prospectively for 4 years. This result is in accordance with Borgonio-Cuadra et al. [17] showing that miR-186-5p was overexpressed in the plasma of early-stage OA patients compared to controls. In silico analysis has suggested that potential pathways regulated by miR-186 could be signaling by PDGF, developmental biology, membrane trafficking, and collagen formation. However, Li et al. [19] showed that miR-186 originated from the synovial membrane and belongs to a panel of seven miRNAs that were significantly upregulated in late-stage OA synovial fluid compared to early-stage irrespective of age, gender, and BMI. The reasons for this discrepancy are not clear but could be the geographic origin of patients, North America vs Europe, or the biological fluids analyzed, synovial fluid vs serum/plasma. Moreover, patients from Li’s study were selected from a group with degenerative meniscal tears undergoing arthroscopic surgery (early OA) or total knee replacement (late-stage OA) while Borgonio-Cuadra et al. [17] and us have selected patients on the radiographic score only, without any consideration regarding the OA etiology.

Together with the serum association of miR-146a-5p and prevalent OA, these data reinforce the notion that the miRNAs involved in OA pathophysiology vary according to the stage of the disease [19]. Finally, miR-186 has been essentially studied in cancer, Alzheimer’s disease, neuropathic disorder, and acute myocardial infarction [41]. It can be noticed that the last 3 diseases have been shown to be linked to OA. Recently, Weber et al. [42] demonstrated an association between OA and the risk of dementia. The miR-186 belonged to a set of 27 differentially expressed miRNAs between patients suffering from Alzheimer’s disease and controls [43]. The pathways potentially downregulated are linked with neuronal synaptic functions. Moreover, neuropathic pain is at least partially responsible for pain in OA [44]. Interestingly, miR-186 negatively regulated CXCL13, also known as B lymphocyte chemoattractant, and its downregulation in spinal neurons after spinal nerve ligation causes upregulation of CLCX13 to drive neuropathic pain [45]. Finally, several cross-sectional studies have shown a strong association between OA and cardiovascular diseases [46], and the overexpression of miR-186 in plasma have the potential to be used for the early detection of myocardial infarction [47]. However, for the present analysis, no woman suffered from dementia. Moreover, we had incomplete information regarding cardiovascular events, and we did not have data concerning pain in the OFELY study. So, the potential links between miR-186-5p, cardiovascular diseases, dementia, and OA could not be evaluated.

Our study has strengths and limitations. We investigated the association between the serum miRNAs expression and prevalence and for the first time incidence of knee OA in a well-characterized population of women followed prospectively over a long period of time of 4 years. We have chosen to carry out the validation step of our study by analysis of duplicate samples on TaqMan microRNA arrays, in order to reduce erratic manipulations and improve the reproducibility between the samples. Moreover, our study combines the validation of our NGS results and the first attempt to replicate previously published results, a process that had been lacking in the miRNA research field. Finally, we have indicated that women selected for the analyses of prevalent and incident knee OA had also OA at other sites (Table 1). This is a key point because we showed in the OFELY cohort that at this mean age, 68 ± 8 years, the prevalence of OA was high with 75% and 88% of women having osteophytosis at the lumbar spine and thoracic spine, respectively [48, 49] in agreement with previous prevalence data [50, 51]. Consequently, in both groups, knee OA and healthy women, we have verified that the presence of OA at other sites did not cause any bias between the groups (see Table 1) in order to decrease as much as possible the effects of this unavoidable situation of generalized OA on the detection of miRNAs associated with knee OA. However, we studied knee OA in women only, and the number of participants is limited. It has been demonstrated that gender may influence the level of the expression of miRNAs [52]. Consequently, our results need to be confirmed by additional larger studies and particularly in males. It can be noticed that miR-146 and miR-186 have been quantified in serum patients because the miR-3p forms of both miRNAs were almost undetectable in the 20 tested sera in the screening phase (data not shown). This lack of detection in the serum is probably related to thermodynamic instability of the 3p arms and to their subsequent degradation in the cellular p-bodies. Finally, the molecular mechanisms associating serum miR-146 and miR-186 to OA remain to be further clarified, for example, the role of the single-nucleotide polymorphisms in the promoter region of miR-146 gene [53].

Collectively, our results show that miR-146a-5p is increased in women with mild to moderate knee OA compared to healthy women. Importantly, miR-186-5p is also increased in those women who will develop radiographic knee OA over the next 4 years; therefore, this miRNA has the potential to detect preclinical knee OA.