Tissue miRNAs have been noted not only as key molecules in intracellular regulatory networks for gene expression, but also as biomarkers for various pathological conditions [26
]. Recent studies suggest that miRNAs in plasma can be biomarkers for the diagnosis of lung, colorectal and prostate cancer [14
]. Plasma miRNAs are also suggested to be potential biomarkers for drug-induced liver injury, and myocardial injury [28
]. In this report, we showed the presence and the stability of miRNAs in synovial fluid and plasma. We also found that the expression of miRNAs in synovial fluid was distinct from that in plasma and may reflect the condition of joint space. Consistently, synovial fluid concentrations of miR-16, miR-146a, miR-155 and miR-223 were significantly higher in RA than those in OA. Finally we referred the possibility of plasma and synovial fluid miRNAs as potential biomarkers of RA.
We quantified miRNAs by real-time PCR after using NCode VILO miRNA cDNA Synthesis Kit. This kit polyadenylates miRNAs and reverse-transcribes with a poly(T) adapter as reverse primer. Because the specificity of this procedure depends on the annealing of the forward primer to the sequence of mature miRNA in the amplicon during amplification, there is a low possibility that pre-miRNAs are also amplified [30
]. To exclude the contamination of pre-miRNAs and nonspecific amplification, we performed TA cloning of PCR products. We verified that all the inserted size was approximately 60 nucleotides by electrophoresis, and that sequences were correct. These results were probably attributed to low abundance of pre-miRNAs and difficulties in polyadenylation of pre-miRNA due to the presence of the stem loop structure [31
]. Even if there remains little possibility to amplify pre-miRNA, we think that procedures used in this study are useful for diagnosis and determination of activities.
Plasma miRNAs have been shown to be remarkably stable in plasma and protected from endogenous RNase activity [14
]. In previous reports, plasma miRNAs are stable at room temperature for up to 24 h and resistant for freeze-thawing from -80°C to room temperature up to eight times. We additionally demonstrated that miRNAs in synovial fluid were as stable as miRNAs in plasma and that both of these miRNAs were also stable at -20°C for up to seven days. These stabilities contribute to the handiness of plasma and synovial fluid miRNAs as biomarkers.
Although we showed that synovial tissue is a main source of synovial fluid miRNA, the mechanism for stability of synovial fluid miRNA remains to be determined. In plasma, some miRNAs are thought to be secreted in a form of exosomes, which are 50- to 90-nm membrane vesicles abundant in plasma containing mRNAs and miRNAs [32
]. Exosomes released from various cells can transfer proteins and RNA between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells [34
]. However, other mechanisms for stabilization may exist (for example, in a RNA-induced silencing complex (RISC)), because some miRNAs were reported to be biomarkers of tissue injury (for example, liver, heart, kidney, et al
.). Exosomes were shown to exist in synovial fluid [38
], but there have been no report about the existence of miRNA in synovial fluid or its exosomes.
Investigated miRNAs in this study have already been shown to associate with RA or OA. miR-16 and miR-132 were shown to be upregulated in PB MNCs of RA patients [21
]. Although the function of miR-16 and miR-132 in RA has not been determined yet, miR-16 is present in high levels in most of cells and thought to be potentially a master miRNA
involved in determining mRNA stability via AU-rich element sites [39
]. miR-146a is upregulated in PB MNCs, FLS and synovial tissue of RA [19
] and expressed in cartilage of low-grade OA [40
]. The targets of miR-146a/b are IL-1β and TRAF6, which is a key molecule in the down stream of TNFα and IL-1β signaling [41
]. The expression of miR-155 is upregulated in RA FLS and has repressive effect to MMP-3 and 1 [18
]. The expression of miR-223 is down regulated in RA FLS [19
Our hypothesis was that in RA patients, miR-16, miR-132, miR-146 and miR-155 were upregulated in plasma and synovial fluid, but miR-223 down regulated. However, there were no statistically significant differences between plasma miRNAs of RA and those of OA. These results are not inconsistent with the previous report: Expression patterns of exosomal miRNAs were shown to be different from those of intracellular miRNAs [34
], though we could not directly show that the synovial fluid miRNA exist in the form of exosome. We showed synovial fluid miRNAs were similar to miRNAs secreted by synovial tissues, while plasma miRNAs were different from miRNAs secreted by MNCs. These facts suggest that synovial tissues and infiltrating cells are a main source of synovial fluid miRNAs, while plasma miRNAs are generated by various tissues.
In this study, all healthy controls were younger than 66 years old according to the request of our ethical committee, while patients with OA were older than 64 years old. When the age of patients and healthy controls was limited from 40 to 60 years to match the age background of groups, plasma miR-132 of HC (n = 9) was still significantly higher than that of RA (n = 16) (P < 0.01). This result suggests that the difference in age between groups has little effect on our analyses.
Plasma concentration of miR-132 differentiated patients with RA or OA from HC, though plasma and synovial fluid miR-132 failed to differentiate RA from OA. Furthermore, plasma miR-132 or its SF/PB ratio correlated with TJC. These results indicate that miR-132 might be involved in the systematic condition of patients with joint inflammation.
On the other hand, miR-16, miR-146a, miR-155 and miR-223 were higher in RA synovial fluids than in OA synovial fluids. Although these miRNAs of plasma had no differences between RA and OA, they significantly correlated with TJC, and plasma miR-16 also correlated with DAS28. Moreover, SF/PB ratio of miR-16 and miR-146a also correlated with TJC with moderate R2 values. These collectively imply that miR-16, miR-146a miR-155 and miR-223 are involved in the pathogenesis specific for RA.
As reported in the field of malignant tumors [14
], disease specific plasma miRNAs for RA or OA are expected. Although investigated plasma miRNAs failed to differentiate RA and OA, disease specific miRNAs that are not investigated in this study may exist. In our preliminary study, miR-124a, miR-142-3p, miR-142-5p, and miR-133a were also detectable. Further analysis for comprehensive plasma and synovial fluid miRNAs using larger number of samples including age-matched RA and OA patients with various severity and healthy controls are expected.