Familial Mediterranean fever-related miR-197-3p targets IL1R1 gene and modulates inflammation in monocytes and synovial fibroblasts

Familial Mediterranean fever (FMF); is an autosomal recessively inherited autoinflammatory disease caused by the mutations in the Mediterranean Fever (MEFV) gene. Recent studies have shown that epigenetic control mechanisms, particularly non-coding RNAs, may play a role in the pathogenesis of autoinflammation. microRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating host gene expression at the post-transcriptional level. The phenotypic heterogeneity of FMF disease suggests that FMF may not be a monogenic disease, suggesting that epigenetic factors may affect phenotypic presentation. Here we examined the potential anti-inflammatory effect of miR-197-3p, which is a differentially expressed miRNA in FMF patients, by using inflammation related functional assays. We monitored gene expression levels of important cytokines, as well as performed functional studies on IL-1β secretion, caspase-1 activation, apoptosis assay, and cell migration assay. These experiments were used to evaluate the different stages of inflammation following pre-miR-197 transfection. Anti-miR-197 transfections were performed to test the opposite effect. 3′UTR luciferase activity assay was used for target gene studies. Our results obtained by inflammation-related functional assays demonstrated an anti-inflammatory effect of miR-197-3p in different cell types (synovial fibroblasts, monocytes, macrophages). 3′UTR luciferase activity assay showed that miR-197-3p directly binds to the interleukin-1beta (IL-1β) receptor, type I (IL1R1) gene, which is one of the key molecules of the inflammatory pathways. This study may contribute to understand the role of miR-197-3p in autoinflammation process. Defining the critical miRNAs may guide the medical community in a more personalized medicine in autoinflammatory diseases.

example, 15-18% of patients with clinical diagnosis of FMF do not have identified pathogenic variant. Furthermore the "variants of uncertain significance" have different clinical effects in patients 8 . Individuals who have the same genotype, but different phenotypes, may have modifying genes, epigenetic factors, or the environment which could influence the genotype-phenotype relationship seen in FMF.
DNA methylation, histone modifications, and noncoding RNAs (ncRNAs) can be considered as main epigenetic control mechanisms. ncRNAs are classified as small or long, according to the number of nucleotides present in the RNA molecule. microRNAs (miRNAs) are a well-known group of small ncRNAs, that may have a role in the phenotypic heterogeneity observed in genetic diseases. miRNAs are typically 20 to 25 nucleotides in length, do not encode proteins and regulate gene expression. About 2656 mature miRNAs (MiRBase, https ://www.mirba se.org, release 22) have been identified in humans and are thought to regulate 60% of proteinencoding gene expression 9 . A single miRNA can regulate the expression of dozens of genes by inhibiting protein translation or degrading mRNA 10 .
miRNA expression patterns of each cell type, contribute to the formation of tissue specific properties and functions. miRNAs can alter gene expression within their own genes and act as intercellular communication molecules. Recent studies have shown that miRNAs can be packaged in exosomes or microparticles, then released from cells into surrounding tissue or circulation [11][12][13] . Because of these unique features (disease specificity, high stability and accessibility), miRNAs are emerging as important clinical biomarkers useful for the diagnosis of specific diseases and for monitoring treatment responses 14 .
In recent years, many studies have determined that miRNAs may be involved in the regulation of inflammatory processes 15 . miRNAs have been identified in the pathways associated with the immune response, such as the development and differentiation of B and T cells, proliferation of monocytes and neutrophils, activation of the antibody and release of inflammatory regulators 16 . Many common miRNAs have been associated with multiple diseases, such as miR-155 and miR-146a in rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and bacterial infections 15 . miR-21 is a tributing factor to the pathogenesis of many autoimmune diseases (type I diabetes, psoriasis, MS, SLE, Sjogren syndrome (SS)) and may play an important role in the regulation of autoimmune responses [17][18][19][20] .
Several studies have identified miRNAs that may be associated with FMF 21 . These studies focus on the levels of miRNAs without the aid of functional data. Wada et al. 22 showed that the levels of circulating miRNAs changed during FMF attacks in patients within distinct FMF subgroups, due to specific mutations in the MEFV gene. Latsoudis et al. 23 found a significant increase in the expression of miR-4520a following the silencing of the MEFV gene in human pre-monocytic cell line (THP-1) cells. Functional assays showed that this miRNA was bound to the gene which has a Ras homologue enriched in brain (RHEB) in the brain. The target of RHEB was the main activator in the mammalian target of rapamycin (mTOR) signaling pathway of rapamycin. Analysis of blood from ten p.M694V homozygous FMF patients showed increased expression of miR-144-3p, miR-21-5p, miR-4454 and miR-451a in FMF patients compared to healthy controls, while the expression of miR-107, let-7d-5p and miR-148b-3p decreased 24 . miRNA analysis performed in 51 patients, as well as 49 healthy controls, to investigate the effect of miRNAs on the pathogenesis of FMF 25 . Koga et al. 26 investigated serum samples of FMF patients with and without FMF attacks. The expression of miR-204-3p decreased in the sera of FMF patients. miR-204-3p inhibited inflammatory cytokine release via phosphoinositide 3-kinase gamma (PI3Kγ) pathway and could be used as a good biomarker.
In the presented study, we examined the effect of miR-197-3p on synovial fibroblasts, monocytes, macrophages. We identified this miRNA as one of the differentially expressed miRNAs in whole blood samples of FMF patients 27 and in inflammation in different cell lines. miR-197-3p levels had been reported in different cancers including p53-dependent lung cancer 28 , non-small cell lung cancer 29 , and hepatocellular carcinoma 30 . miR-197-3p decreased in FMF patients therefore our first experiments were performed with pre-miR-197 transfections to increase the expression level of this miRNA. We have analyzed gene expression levels of inflammatory cytokines and performed functional experiments on IL-1β secretion, caspase-1 activation, apoptosis, and cell migration assays to test the different stages of inflammation following pre-miR-197 transfection. We observed a significant decrease in inflammation parameters in miR-197-3p overexpressing cells. To test the opposite effect, anti-miR-197 transfections were done and it was found that inflammatory genes expression levels and IL-1β secretion significantly increased as expected. Next, we performed candidate gene studies with a 3′ UTR luciferase activity assay and demonstrated that miR-197-3p targets IL1R1. These data strongly suggest the potential antiinflammatory effect of miR-197-3p and its possible influence on the autoinflammation.

Results
miR-197-3p modulates the gene expression level of IL-1β, MEFV, IL-18, TNF-α, and TGF-β in SW982, THP-1 cells, and THP-1 derived macrophages. In our previous study, miR-197-3p is significantly decreased in homozygous (M694V/M694V) FMF patients compared to controls. This was shown in both miRNA microarray and qRT-PCR analysis 27 . In order to understand the possible anti-inflammatory effect of miR-197-3p, we transfected SW982 cells with pre-miR-197 and checked the expression of inflammatory cytokines and MEFV by qPCR. The expression levels of IL-1β and MEFV genes were decreased in pre-miR-197 transfected cells. TGF-β and TNF-α expression levels were also non-significantly decreased in pre-miR-197 transfected cells, while IL-18 expression level was significantly increased (Fig. 1a). We have shown that after transfection of pre-miR-197, the expression level of proinflammatory cytokines and transcription factors decreased significantly by targeting pre-miR-197′s potential target genes to suppress inflammation. Expression analysis supports that this miRNA may be involved in pathways such as cytokine release and stimulation of inflammation through its target gene. Suppressing the target gene suppresses inflammation resulting in an anti-inflammatory effect. In THP-1 cells, the expression levels of IL-1β, MEFV, TNF-α, and TGF-β genes were decreased in transfected cells while www.nature.com/scientificreports/ IL-18 gene expression was increased in a statistically non-significant manner (Fig. 1b). These results were like the findings in SW982. We next investigated the endogenous expression of miR-197-3p following TNF-α and TGF-β stimulation. TNF-α treatment significantly increased the level of miR-197-3p expression, whereas there is no difference in miR-197-3p expression level after TGF-β treatment (Fig. 1c). The increase in miR-197-3p expression following TNF-α stimulation, may be explained with its possible function in suppressing inflammation as an anti-inflammatory molecule. IL-1β gene expression level in THP-1 derived macrophages was increased after anti-miR-197 transfection and decreased significantly with pre-miR-197 transfection (Fig. 2a). The same pattern was seen in MEFV and IL1R1 genes (Fig. 2b,c). But, IL-18 gene expression level decreased with anti-miR-197 transfection. This result indicated that IL-18 production may not be related with miR-197-3p, although the gene level was decreased with pre-miR-197 transfection (Fig. 2d).
THP-1 derived macrophages in the presence of LPS showed increase in IL-1β , MEFV, and IL-18 gene expression levels and increased at 48 h and 72 h after anti-miR-197 transfection ( Supplementary Fig. 1a-c). Only IL-1β gene expression level was decreased after 48 h (significantly) and 72 h followed by pre-miR-197 transfection ( Supplementary Fig. 1a). There was no difference in MEFV gene expression level after pre-miR-197 transfection ( Supplementary Fig. 1b). IL-18 gene expression levels decreased after 48 h of pre-miR-197 transfection, but this was not continued to 72 h where there was an increase in the gene expression level ( Supplementary Fig. 1c). These results indicated that IL-1β is the most influenced gene by the miR-197-3p transfections in the SW982, THP-1 cells, and THP-1 derived macrophages.

miR-197-3p suppresses cell migration in SW982 and THP-1 cells. Both SW982 and THP1 cells,
with pre-miR-197 transfection, showed a significant decrease in cell migration at 6 and 12 h, while there was no significant difference with the scramble/mimic controls (Fig. 3a,b). The increase in the expression of pre-miR-197 was accompanied by a decrease in the number of cells migrating under the filter. This decrease in cell migration in pre-miR-197 transfected cells, suggests that this miRNA may influence this pathway or reduce cytokine levels. Thus, downregulation of pro-inflammatory cytokines prevents the activation of signaling pathways related with cell migration in pre-miR-197 transfected cells.

miR-197-3p suppresses caspase-1 activation and apoptosis in SW982 and THP-1 cells.
In pre-miR-197 transfected SW982 cells, caspase-1 activity was significantly lower, compared to THP-1 cells, where there was non-significant decrease in caspase-1 activity. (Fig. 4a). This decrease in caspase-1 activity in transfected cells supported the concept that this miRNA may have an anti-inflammatory effect. Cells become activated and migrated in response to inflammation signals, to sites of inflammation, where they undergo pyroptosis or apoptosis. Apoptosis was tested as another indicator of cell death shown in inflammation in our study. The cell death rate decreased in pre-miR-197 transfected SW982 cells although in THP-1 cells there was no difference (Fig. 4b).
miR-197-3p targets IL1R1 in THP-1 cells. We used the THP-1 cell line for miRNA binding analysis, which naturally expresses pyrin and related proteins. miR-197-3p and its possible target genes were determined by TargetScanHuman (Release 7.2: March 2018). From the given list, IL1R1 was chosen as a candidate target gene according to score, number of 3P-seq tags and its possible role in inflammation process. We next examined expression of IL1R1 in pre-miR-197 transfected THP-1 cells. After pre-miR-197 transfection, IL1R1 gene expression was significantly reduced (Fig. 5a). We sought to analyze a direct interaction of miR-197-3p and IL1R1 by using 3′UTR luciferase activity assay. This was accomplished with a mutation in the IL1R1 gene 3′ UTR, which abrogated miRNAs binding to the locus (Fig. 5b). Following validation of mutation; control, wild type and mutant vectors were transfected into THP-1 cells with miRNAs and negative controls. According to the results of 3′UTR luciferase activity assay: miR-197-3p binds to the gene and luciferase signal decreases when there is no mutation in 3′UTR region of IL1R1 gene. In the case of mutations in this region, the binding region of miRNAs www.nature.com/scientificreports/ disappears and luciferase signal increases (Fig. 5c). Therefore, miR-197-3p has been shown to directly bind to the 3′UTR region of the IL1R1 gene experimentally.

Discussion
Autoinflammatory diseases are caused by dysregulations in the innate immunity, often due to single gene defects 31 . Recent studies have shown that epigenetic control mechanisms, particularly non-coding RNAs, may play a role in the pathogenesis of autoinflammatory and autoimmune diseases. miRNAs, a group of non-coding RNAs have been shown to have activating or suppressing properties in the inflammatory response process. It was found that miRNAs mostly regulate the inflammation process by changing the expression levels of cytokines, transcription factors, and interleukin-related genes 15,16 . In this context, the possible effects of miRNAs on the increased inflammation process in the pathogenesis of FMF were investigated. Considering the mechanism of inflammation in these diseases, the affected cell quickly regains its homeostasis or goes to pyroptosis by passing through several steps. Alterations in these steps eventually lead to a return to physiological state or a delay or suspension of apoptosis 32,33 . The accumulation of components in the inflammatory region, cell migration, caspase activation and infiltration, together with the activation and release of IL-1β, are important events 34 . The effects of specifically miR-197-3p on the inflammation process in this study were investigated in detail by selecting these steps in functional analysis. miR-197-3p was found to have anti-inflammatory effect on inflammation related pathways. miR-197-3p has an inhibitory effect on IL-1β secretion, which is preceded by a reduction in IL-1β mRNA levels. Therefore, it may play important regulatory role in IL-1β depended inflammation processes. It was determined that pre-miR-197 transfection inhibited cell migration, according to filter experiments in synovial fibroblasts, decreased cell migration according to wound healing experiments at 6 and 12 h and decreased apoptotic rate. Another important process associated with inflammation was the decrease in caspase-1 activity.
Functional studies for miR-197-3p utilized a second cell line model, human monocyte cell line (THP-1), in which pyrin and related proteins are naturally expressed. These cells are known to be involved in the inflammation, and thus a good candidate for gene and functional studies. Cell migration was decreased in pre-miR-197 transfected THP-1 cells. IL-1β, MEFV, TGF-β, and TNF-α gene expression levels were decreased as well in relation to inflammation. When considered together, miR-197-3p demonstrated an anti-inflammatory effect in monocytes as well and suppressed inflammation. It is recommended to validate miRNAs' function in at least two different cell lines and determine if similar results are obtained. In this context, similar results obtained in monocytes are very important. For anti-miR-197 transfections, THP-1 derived macrophages were used as a third cell type. Pre-miR-197 transfections were included to the experimental set-ups. Cells shown increased IL-1β secretion after anti-miR-197 transfection as expected. In addition, IL-1β, MEFV, and IL1R1 gene expression levels were www.nature.com/scientificreports/ also increased after transfection. Similar results were obtained from cells triggered with LPS and other inflammasome activators. These results help to clarify the function of miR-197-3p in inflammation.
Caspase-1 dependent cell death occurs as a result of activation of inflammatory complex is called 'pyroptosis' 35 . Cell death experiments with Annexin V / PI staining measure apoptosis, where caspase-1 activity are correlated to pyroptosis, the mode of death in inflammatory cells. Therefore, caspase-1 activity assays were also evaluated for the determination of pyroptosis. According to the results of caspase-1 activity experiments, miR-197-3p was also found to reduce pyroptosis.
It is possible that miRNAs circulating in exosomes, which are therefore conserved in plasma or serum, can be used as potential biomarkers. In another project in which our group is involved, the presence of candidate miRNAs in exosomes isolated from serum samples of FMF patients were investigated and it was observed that the level of miR-197-3p decreased in exosomes of patients compared to controls (Hacettepe University, Noninterventional Clinical Researches Ethics Board, National ethics committee approval has 13.06.2017 date and GO17/513-11 number. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.) (unpublished data). This data obtained in exosomes supports the hypothesis that the decrease in this anti-inflammatory feature of miRNA systemically in patients leads to increased inflammation and affects the pathogenesis.
Considering the microarray results obtained in our previous study, the downregulation of miR-197-3p in severe FMF patients, further supports the anti-inflammatory effect hypothesis 27 . Therefore, the inflammatory process is triggered, or inflammation may not be suppressed due to decreased expression of miR-197-3p in patients. In our previous study, miR-197-3p has shown a significant decrease in adult FMF patients. In this present study, we analyzed the level of miR-197-3p in pediatric FMF patients. miR-197-3p level was reduced significantly in severe FMF and systemic autoinflammatory diseases (SAIDs) patients, compared to mild FMF patients (Supplementary Fig. 2) in pediatric study group (Hacettepe University, Non-interventional Clinical Researches Ethics Board, National ethics committee approval has 02.12.2015 date and GO15/744-19 number). It is thought that the decrease in this miRNA can be seen in FMF in different age groups, has an epigenetic regulation not age-related and is critical for the inflammation seen in FMF. www.nature.com/scientificreports/ The identification of candidate genes of miRNAs is crucial and necessary in order to fully elucidate the functions of miRNAs. Therefore, candidate gene studies for miR-197-3p were performed considering the possible effect on the pathogenesis of FMF. We have shown that the gene target of miR-197-3p was the IL1R1 gene, both by bioinformatic analysis and 3′UTR luciferase activity assay. A direct linkage was experimentally demonstrated, suggesting that miRNA may be involved in the upstream pathways of inflammation. The IL1R1 gene encodes a cytokine receptor of the IL-1 receptor family and the encoded proteins serve as IL-1α and IL-1β receptors. It has an important role in immune and inflammatory response regulated by many cytokines 36 . miR-197-3p reduces the number of receptors located on the membrane by suppressing IL1R1 expression. IL-1β decreases NF-κB stimulation mediated by signal transduction and consequently, the precursor IL-1β (pro-IL-1β) expression decreases. Given the pathogenesis of FMF, inflammation from mutant pyrin is thus suppressed to some extent. Hypothetically IL1R1 expression in colchicine-resistant M694V homozygous patients with persistent inflammation may be increased due to decreased miR-197-3p expression. As a result, NF-κB mediated precursor IL-1β expression increases, leading to an increase or inability to suppress existing inflammation.
Our results provide insight to the mechanisms underlying autoinflammatory diseases, the epigenetic regulation with miRNAs in terms of these diseases and may contribute to the development of new diagnostic (biomarker) and treatment strategies. Our results revealed the anti-inflammatory effect of miR-197-3p. As miR-197-3p expression was decreased in our patients, this may explain the severe phenotype that was seen in these individuals. In this case, it is predicted that by increasing miR-197-3p expression in patients, an inflammationsuppressing effect can be achieved. Different target genes related with autoinflammation may exist for this miRNA, yet these genes may help us to understand the role of miR-197-3p in inflammation. Although many autoinflammatory diseases are rare, a better understanding of their pathogenesis will help to understand the basic mechanisms of inflammation and innate immunity, which are important for common diseases. By investigating the factors that cause such diseases, new effective and preventive treatment protocols can be developed.   Wound healing assay. SW982 cells were seeded in six-well cell plate at 50% of confluency and transfected with 20 nM pre-miR-197 or scramble/mimic control. After transfection, the cells were expected to reach 90% confluency and then two scratches were made by sterile 10 μl pipette tip. The culture was photographed by Leica IM50 fluorescence inverted microscope at 0, 6, 12 and 24 h.

Methods
Migration assay. Migration assay was performed by the Transwell migration chamber. Polycarbonate membrane filters containing 8 µm pores were placed in 24-well cell culture plate. The filters used in this experimental system have two compartments, namely the upper and lower wells. Firstly, the medium was placed under and above the filter and incubated for 1 h at 37 °C in 5% CO 2 incubator. Cells transfected with pre-miR-197 or scramble/mimic control for 48 h were harvested and resuspended in serum-free medium. Transfected cells were then transferred to the upper chamber at a density of 3 × 10 4 cells/well. After 24 h, non-migrated cells were removed by cotton swap. The cells remaining at the bottom of the filter were treated with 1 mM calcein-AM and incubated for 30 min in at 37 °C in 5% CO 2 incubator. At the end of the incubation, three pictures were taken from the lower area of the filter with the Leica IM50 fluorescence microscope.
Caspase-1 activity assay. Caspase-1 activity was analysed in pre-miR-197 or scramble/mimic control transfected cells using the Caspase-1 Fluorometric Assay Kit (Abcam) according to the manufacturer's guidelines. Following the protocol, the samples were measured at Ex/Em = 400/505 nm using SpectraMax M2 microplate reader. www.nature.com/scientificreports/ Biosystem) PCR. IL1R1 3′UTR / mut IL1R1 3′UTR reporter constructs were co-transfected into THP-1 cells together with pre-miR-197 miRNA precursor or scramble/mimic control. At 24 h of post-transfection, luciferase activities were analyzed using the LightSwitch Luciferase Assay Reagent (SwitchGear Genomics). These assays are performed by using a pLightSwitch luciferase construct containing the full-length wild-type target gene 3′UTR or its mutant version. This provides functional evidence for direct binding of a miRNA specific to 3′UTR in a cell-based system. The luciferase signal from the cells was measured by Molecular Devices, Spectramax i3x microplate reader.