Pathogenesis of Rheumatoid Arthritis

#### **Chapter 1**

## Pathogenesis, Pathology and Genetics of Osteoarthritis

*Ferhat Ege*

#### **Abstract**

Osteoarthritis (OA) is a condition with high prevalence worldwide. OA affects not only the articular cartilage, but the entire joint, including the subchondral bone, ligaments, capsule, synovial membrane and the periarticular muscles. Despite the fact that the risks associated with OA increase with age, it is not a part of the natural aging process. It typically involves the knee, hip, spine, hand and foot joints. Several factors play an important role in the pathogenesis of OA, including biomechanical factors, proinflammatory mediators and proteases. On the other hand, it was mostly the results of the studies conducted on the genetic, genomic and epigenetic aspects of OA, from among many of its underlying etiological factors, which shed light on the molecular processes involved in the etiopathogenesis of OA. As the mechanisms that cause joint tissue damage in OA come to light, the treatment of OA will go beyond just providing symptomatic relief. Consequentially, new treatments will emerge that will either slow or completely stop the progression of OA.

**Keywords:** Osteoarthritis, Genetics, Epigenetics, Etiopathogenesis, Pathology

#### **1. Introduction**

Osteoarthritis (OA) is a chronic disease that affects all structures of the joint as well as the periarticular tissues. In the past, OA was considered simply as a degenerative joint disease, yet the pathogenesis of OA is in fact much more complex than just wear and tear. Hence, the term "osteoarthritis" is indeed a pertinent term, as the suffix "itis" is indicative of an inflammatory process. It is estimated that approximately 50% of the world population over 65 years of age is affected by OA. The symptomatic treatment of this common disease provides regression of symptoms, nevertheless it often does not constitute an effective treatment option thus causing an increase in the OA-related health expenditures. The elucidation of the etiopathogenesis of OA and the molecular studies to be carried out in respect thereof are likely to allow early diagnosis of OA and also contribute to the development of new treatment options.

#### **2. Pathology and pathogenesis of osteoarthritis**

Articular cartilage degeneration, which develop as a result of the deterioration of the balance between the production and destruction of cartilage, new bone formation, sclerosis of subchondral bone, ligament and meniscus damage, periarticular muscle weakness, synovial inflammation and fibrosis are all involved in the pathogenesis of OA [1]. Hence, the pathogenesis of OA would be better understood provided that the structure of the joint and the related histopathological features are reviewed.

#### **2.1 Structure of the joint**

Synovial joints consist of an articular cartilage that covers the ends of the opposing bones, the synovial fluid that nourishes and lubricates the tissues, the synovium that secretes the synovial fluid, the ligaments that hold the skeletal elements together, the tendons that connect the bones with the muscles, and the joint capsule surrounding the joint. In order to have normal joint functions, it is necessary that the opposing joint surfaces move over each other painlessly, that the load on the joint tissues is homogeneously distributed, and that the stability to that effect is sustained [2].

Articular cartilage is a connective tissue located at the bone ends and which has a thickness of 0.2 mm to 6 mm depending on the location. Articular cartilage provides a smooth and low-friction surface that primarily allows for normal gliding motion of the articular surfaces [3]. Cartilage consists of an extracellular matrix, 65–80% of which is water and 20–35% of which is solid matter, and of chondrocytes dispersed in this matrix. 5–6% of the tissue is composed of inorganic material consisting mostly of hydroxyapatite. The organic matter on the other hand is composed of fibrous proteins (collagen), hydrophilic sulfated proteoglycans (chondroitin sulfate, keratan sulfate I and II) and unsulfated proteins (hyaluronic acid). 90% of the collagen is type II collagen, whereas the remaining collagen consists of smaller amounts of type IX, XI, III, VI, XII and XIV collagen [4].

A proteoglycan consists of a protein and glycosaminoglycan chains attached to this protein. The most abundant type of proteoglycan is 'aggrecan' [5]. Type II collagen plays a role in maintaining the volume and shape of the content it is part of, whereas proteoglycans play a role in maintaining the hardness and elasticity [6].

Hyaluronic acid is the substance that maintains the viscosity in synovial fluid. Nonetheless, it requires the presence of a large mucinous protein, which is called lubrisin (proteoglycan-4), in order to maintain a low-friction environment and protect the surface of the joint [7].

Articular cartilage is a avascular heterogeneous structure with four different layers which has no nerve innervation and is fed by a bidirectional diffusion system. These layers are the superficial zone, transitional zone, deep zone and calcified zone. The calcified line between the deep zone and the calcified zone is called the Tide mark [8].

The extracellular matrix is synthesized by chondrocytes. Chondrocytes synthesize cartilage matrix molecules and the metalloproteinases which breakdown the matrix. The cartilage metabolism is based on the balance between the anabolic processes and the catabolic processes carried out by the matrix metalloproteinases (collagenase, gelatinase, stromelysin, cathepsin B and D) and the adamalysins [a disintegrin and metalloproteinase (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), aggrecanase] [5]. This balance is regulated by anabolic cytokines such as transforming growth factor beta (TGF-β), insulin-like growth factor-1 (IGF-1) and bone morphogenetic proteins (BMPs) and catabolic cytokines such as interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) [6].

The synovium, which produces the synovial fluid, consists of two layers as inner and outer layers. It is firmly attached to the joint capsule and prevents the synovial fluid from leaving the joint. The inner and outer layers of the synovium are composed of the synovial membrane and fibrous connective tissue, respectively. The

inner layer includes two types of cells. Type A synoviocytes have the characteristics of macrophages, whereas type B synoviocytes are cells with proliferative capacity and produce hyaluronic acid, collagen, lubricin and fibronectin [9].

The joint capsule is a tissue that contains vascular and nervous tissues and is rich in collagen fibers. It protects the whole joint both passively by restricting the movements of the joint and actively through the proprioceptive sensation triggered by the nerve endings.

#### **2.2 Pathological changes that occur in connection with osteoarthritis**

#### *2.2.1 Changes that occur in the articular cartilage*

Chondrocytes are active cells that maintain cartilage through normal anabolic/ catabolic activities. The earliest pathological changes observed in association with OA are the fibrillations seen on the surface of the cartilage. Fibrillations are more common at parts of the cartilage exposed to higher loads. Loosening of the collagen network and loss of aggregate occurs in the cartilage at the onset of OA. This loosening of the collagen network allows the hydrophilic proteoglycans to attract water and expand.

The activity of chondrocytes, the only cell type found in cartilage, accelerates significantly as OA develops, that is, chondrocytes begin to proliferate moderately. Nevertheless, the reasons that trigger this premature aging and changes in the chondrocyte cycle such as inflammation, proteoglycan loss, collagen degeneration and chondrocyte failure, as well as the order of occurrence of these changes are still not fully known [10].

As OA progresses, extensive matrix breakdown and loss occur due to the continued production of the proteases driven by proinflammatory cytokines. Fragmented matrix proteins give rise to the further production of cytokine and protease by chondrocytes through autocrine and paracrine stimulations. Cartilage has limited regeneration capacity, hence once collagen is broken down and lost, regeneration does not occur at a measurable degree [11].

There are various histopathological staging-grading systems that are used to categorize the changes associated with OA according to their severity, extent or order of occurrence. These classification systems classically address the changes that occur in articular cartilage, since OA primarily targets the articular cartilage. One of these systems, the histological evaluation system proposed by **Osteoarthritis Research Society International (OARSI)** is a grading, staging and a scoring system. The grades used in the said OARSI system to classify the changes occur in articular cartilage, key features of these grades and the associated criteria in terms of tissue reactions are shown in **Table 1** [12].

#### *2.2.2 Changes that occur in the bone*

Thickening of the subchondral bone (bone sclerosis) occurs due to increased production of improperly mineralized collagen. Osteophytes occur at the margins of the joints, usually at the insertion sites of tendons or ligaments. Osteophytes seen in the distal interphalangeal joints of the hand are called "Heberden's nodes", whereas the osteophytes seen in the proximal interphalangeal joints are called "Bouchard's nodes". Bone cysts form in the advanced stages of the disease, but bone erosions are not typically seen. Erosive OA is commonly seen in the distal joints of the hands (distal interphalangeals and proximal interphalangeals) and central erosions are also seen as opposed to the marginal erosions seen in rheumatoid arthritis (RA) and gout [13].

*Rheumatoid Arthritis*


#### **Table 1.**

*A cartilage histopathology grading methodology.*

#### *2.2.3 Changes that occur in the synovium*

Four patterns have been described in OA-related synovial pathology, which are hyperplastic, inflammatory, fibrotic and detritic patterns. Hyperplastic pattern is the most common manifestation in all stages of OA. Hyperplastic pattern is considered as an early OA finding in its isolated form. Inflammatory pattern is seen equally in both the early and late stages of OA. Inflammatory cell density in the inflammatory pattern is not as much as it is in rheumatoid arthritis. Fibrotic pattern is characterized by capsular fibrosis reflecting late-stage OA. Detrital pattern is characterized by macromolecular cartilages and debris within the synovium, and reflects late-stage OA [14].

#### *2.2.4 Changes that occur in the meniscus*

The changes that occur in the meniscus in connection with OA are first observed in the medial part of the meniscus. Meniscal tears are both a cause and effect of OA. Meniscal tears further increase the matrix degeneration through the inflammatory mediators which emerge as a result of the damage to the meniscus and may lead to the development of OA [15]. The regeneration capacity of the meniscus is limited. The red zone of the meniscus, which is peripherally located, is the area with the best blood supply and the best regeneration capacity, whereas the white zone of the

meniscus, which is more centrally located, is largely avascular and its regeneration is very slow and inadequate [16].

#### **2.3 Etiopathogenesis of osteoarthritis**

OA refers to a dynamic process, which is triggered by various biochemical and mechanical factors and in which destruction and regeneration both take place. In the past, OA was thought to be a degenerative joint disease that emerged with aging. Yet, it is known today that various factors such as biomechanical factors, proinflammatory mediators and proteases play a role in the pathogenesis of OA [17]. The release of biomarkers indicates that the findings that emerge in the earliest detectable stage of knee OA are bone and cartilage metabolisms that are impaired as a result inflammation [18].

#### *2.3.1 Factors involved in the etiopathogenesis of osteoarthritis*

#### *2.3.1.1 Inflammation*

The number of proinflammatory mediators included in the synovial fluid and tissue and which play a role in OA and is increasing by the day. Early studies on OA were focused on interleukin-1 (IL1), which stimulates cartilage catabolic activity. Nevertheless, the role of IL1 in OA has been questioned over the years, since the IL1 levels in OA joints are much lower than the levels that cause cartilage deterioration. It has been shown in the relevant clinical studies that the inhibition of IL1 in knee [19] and hand OA [20] have not improved the structure and symptoms of the disease.

Cytokines such as IL6, interferon-gamma inducible-protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1) and monokine induced by gamma interferon (MIG) were found to be more abundant in OA synovial fluid than IL1 or TNF-α [21]. This finding suggests that these proinflammatory cytokines play a role in inflammation. It has been demonstrated in experimental animal models that there may be a relationship between IL-6 level and increased cartilage loss. The results of all these studies support the hypothesis that IL-6, as a regulatory cytokine, plays a role in the development of OA [22]. Other cytokines and chemokines involved in cartilage degeneration caused by inhibition of the anabolic process and induction of the catabolic process are IL-7, IL-15, IL-17, IL-18, oncostatin M (OSM), growth related oncogene-alpha (GRO-alpha), chemokine (C-C motif) ligand 19 (CCL19) and macrophage inflammatory protein-1beta (MIP-1beta) [23].

It was demonstrated in some studies that there is complement activity in OA joints. In one of these studies, which was conducted on mice, it was demonstrated that complement activation was inhibited by gene deletion or pharmacological modulation and that, as a result of this inhibition, the joint is protected from surgery-induced OA [24].

Adipokines secreted by adipose tissue cause cartilage damage by activating the inflammatory cytokines along with the matrix metalloproteinases (MMPs) triggered by the inflammatory cytokines. These adipokines include leptin, adiponectin, visfatin and resistin [25].

Prostaglandin E2 (PGE-2) has been shown to inhibit proteoglycan synthesis and increase matrix degradation. Additionally, it was shown that patients with OA have high levels of PGE-2 in cartilage [26]. Furthermore, leukotriene B4, a strong leukocyte chemotaxis, has been shown to stimulate proinflammatory cytokines in human synovial fluid samples [27].

#### *2.3.1.2 Proteases*

Proteases are mediators that play a primary role in the catabolic process of OA. There are several proteases that have a role in the pathogenesis of OA. These proteases are collagenase-containing matrix metalloproteinases, cathepsin K-containing proteases, and serine-containing proteases. As the proteases degrade collagen, the related catabolic process results in the progression of matrix loss, since the cartilage's response to damaged matrix repair is limited [28].

'Agreccan', the largest proteoglycan, provides cartilage elasticity. The ADAMTS family of enzymes, also called aggrecanases (ADAMTS-4-5), is involved in the early stage of OA degeneration and is responsible for aggrecan degradation [28].

Type-2 collagen, the most abundant type of collagen found in the cartilage tissue, provides cartilage tensile strength. It is broken down by collagenase-containing matrix metalloproteinases. MMP13 is considered to be the main collagenase responsible for cartilage destruction in OA [28].

Aggrecanase-2 (ADAMTS-5) and MMP-13 have an important place in the pathogenesis of OA. The development of specific inhibitors to these proteases in the context of the development of potential modifying treatments for OA has been of interest [29].

#### *2.3.1.3 Molecular patterns associated with cartilage damage*

Damage-associated molecular patterns (DAMPs) are molecules released from the chondrocytes in the damaged cartilage. DAMPs include extracellular matrix proteins, high mobility group box 1 protein (HMGB1), advanced glycation end products (AGEs) and receptor for advanced glycation endproducts (RAGEs), and alarmins [S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9)].

It has been demonstrated that DAMPs have important roles in the pathogenesis of OA. DAMPs activate intercellular signaling pathways such as RAGE, toll-like receptors (TLR) and mitogen-activated protein kinases (MAPKs), thereby inducing the expression of catabolic proteases and inflammation-related genes [30]. DAMPs give rise to the increase in MMPs and activated macrophages which in turn lead to chondrocyte apoptosis and cause damage to the extracellular matrix (ECM) and cartilage [31, 32].

Fragmented matrix proteins such as cartilage oligomeric matrix protein (COMP), fibromodulin, proteoglycan, collagen, tenascin C, fibronectin, biglycan and aggregate are released from the damaged matrix. These fragmented matrix proteins stimulate the immune response. Consequentially, TLR and integrin are activated and the upregulation of the degenerative pathway is achieved [23, 33–35].

RAGE is a member of the immunoglobulin family. It is expressed in chondrocytes and macrophages. RAGE has been demonstrated to increase in OA joints. This increase causes the production of MMPs, which play a direct role in the pathogenesis of OA [36].

Alarmins are intracellular proteins secreted from bone cartilage or synovium in OA. It is an important member of DAMP family that has a role in the pathogenesis of OA [33].

HMGB1 is a nonhistone nuclear protein. Its release from the nucleus is associated with the apoptosis or necrosis or inflammatory stimulation of cells [37]. A significant increase occurs in the secretion of proinflammatory cytokines, chemokines and MMPs with the increase in HMGB1 secretion [38].

S100A8 and S100A9 are secreted from granulocytes, macrophages and monocytes. There is evidence that these proteins play a role in the cartilage damage and OA progression [39]. In addition to their catabolic effect, S100A8 and S100A9 lead to the formation of bone/osteophyte [40].

It has been demonstrated in the literature that the basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals, from among the inorganic calcium crystals, accumulate in the synovial fluid [41]. Calciumcontaining crystals trigger the inflammatory process by either directly stimulating the chondrocytes or indirectly stimulating the immune system [30]. Additionally, it has been reported in the literature that monosodium urate crystals also trigger inflammation and cause cartilage damage [42].

#### *2.3.1.4 Free oxygen radicals*

The amount of free oxygen radicals and the extent of the DNA damage they cause are higher in OA cartilages than in cartilages without OA. Free oxygen radicals have an important place in OA progression, since they increase the synovial inflammation and cartilage destruction [43].

#### *2.3.1.5 Biomechanical factors*

Abnormal mechanical loading has an important role in the onset and progression of OA [44]. Abnormal mechanical loading may be caused by various factors such as obesity, joint alignment disorders or joint instability. Abnormal mechanical loading leads to mechanical damage in the joint and result in an increase in the release of matrix-degrading enzymes. Cartilage destruction products trigger inflammation and damage to the joint cartilage occurs through cytokine activation.

#### **2.4 Genetics of osteoarthritis**

The molecular processes underlying OA, which have a complex etiology, have become clearer through genetic and epigenetic studies. OA has been categorized as early-onset OA and late-onset OA. Genetic factors are more prominent in the earlyonset OA. Genetic studies on the early-onset OA will provide a better understanding of the etiopathogenesis of the disease.

Family and twin studies have been conducted to reveal the genetic factors in OA [45]. To give a few examples, in the family studies conducted by Kellgren in UK and US, it was determined that there is a genetic component of the hand and knee OA [46], whereas in the study conducted by Lanyon et al., it was shown that the risk of radiographic hip OA is higher in the siblings of the patients with advanced hip OA [47].

Studies, in which monozygotic (MZ) and dizygotic (DZ) twins were compared, have shown that genetic factors are effective in OA. In one of these studies, it was shown that genetic factors are 39–65% effective on hand and knee OA radiographs, independently of environmental and demographic factors [48]. In another study, knee OA progression was investigated in 114 MZ and 195 DZ female twin couples. Consequentially, a higher correlation was found in the MZ twins than in the DZ twins in terms of both osteophyte and joint space narrowing, and the heritability was calculated as 62% for osteophyte progression and 72% for joint space narrowing progression. Additionally, it has been reported that the genetic effect on knee OA progression is more prominent in the medial compartment [49]. Furthermore, it has also been reported that the genetic effect differs according to the affected area in OA. Accordingly, the heritability was reported as 40%, 60%, 65% and 70% in the knee, hip, hand and spine regions, respectively [48].

Candidate gene studies have focused on many gene groups such as cartilage structural genes [collagen type II alpha 1 (COL2A1), collagen type IX alpha 3 (COL9A3), collagen type XI alpha 1 (COL11A1)], genes associated with bone mineral density (BMD) [vitamin D receptor (VDR), estrogen receptor 1 (ESR1)], genes associated with chondrocyte cell signal transduction (bone morphogenetic protein 5 (BMP5), frizzled-related protein B (FRZB), interleukin-4 receptor alpha (IL-4Rα)], inflammatory cytokine genes (IL-1, IL-10, TGFβ1, IL-6, TNFα) [50].

The finding that VDR gene polymorphism is associated with BMD lead to the studies on the possible relationship of VDR gene polymorphism with OA [51]. In this context, it was shown in a study conducted on 543 women in Finland that VDR polymorphism plays a role in the etiology of symmetrical hand OA [52].

The prevalence of knee OA is significantly higher in women than in men. This difference was atrributed to the estrogen receptor α (ERα), which is encoded by ESR1. Several polymorphisms in ESR1 [PvuII (rs2234693) and BtgI (rs2228480)] have been confirmed as risk factors for OA [53].

FRZB is a glycoprotein and plays a role in chondrocyte maturation and bone development. In Rotterdam and Genetics, Osteoarthritis and Progression (GARP) studies, R324G single nucleotide polymorphism (SNP) of the FRZB gene was found to be associated with generalized OA, whereas rs7775 and rs2888326 SNPs were found to be associated with knee and hip OA [50]. BMPs are bone-derived factors that can induce new bone formation. In a study conducted by Sharma et al. on BMP5 gene, rs1470527 and rs9382564 polymorphisms were shown to be significantly associated with knee OA [54].

The hypothesis put forward in candidate gene studies is still being investigated in terms of the genetic variant. Researchers favor genome-wide association studies (GWAS), which is a hypothesis-free approach, in the event that they think that candidate gene studies do not contribute much to the etiopathogenesis of the disease. GWAS allows the identification of genetic loci and the discovery of new genetic variants. In this context, GWAS contributes to the discovery of prognostic biomarkers that can contribute to early diagnosis and the identification of new areas that can be targeted by medical treatments [55–58]. The number of OA genetic risk loci, most of which have small effect sizes, has increased to 90 in the GWAS studies carried out up till 2019 [59]. 56 new loci were identified in the two major OA analyzes published recently [59, 60]. First of these two studies, that is the deCODE (Decode Genetics, Iceland)-UKBB (UK Biobank, England) study, was conducted with more than 650,000 British and Icelandic citizens. 11.6 million genotype variants were examined within the scope of the said study, and 23 significant variants were detected in 22 loci [60]. Second of these studies, that is the Arthritis Research UK Osteoarthritis Genetics (arcOGEN)-UKBB study, was conducted with more than 455,000 British citizens. 17.5 million genotype variants were examined within the scope of the said study, and 65 significant variants were detected in 64 loci [61]. These studies, which were conducted via performing separate meta-analyses for the hip and knee OAs, are the largest OA GWAS studies published to date (**Figure 1**) [59].

Genetic variations are grouped into single nucleotide substitutions (mutations and single nucleotide polymorphisms (SNPs)], insertions and deletions, copy number variations or short tandem repeats [62]. Variations in the genome underlie the differences between the individuals. The most common of these variations are SNPs. SNPs are considered to be associated with susceptibility to diseases [63]. The majority of the common diseases that give rise to SNPs, including OA, are considered to affect the transcription of nearby genes by altering the transcription factor binding [59].

Epigenetics plays an important role in the regulation of gene expression and is associated with the pathogenesis of a number of human diseases. The term

*Pathogenesis, Pathology and Genetics of Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.99238*

#### **Figure 1.**

*The new OA risk loci identified in either or/both of the deCODE-UKBB and arcOGEN-UKBB studies.*

epigenetics encompasses DNA and chromatin modifications and the functions related thereto, in addition to non-coding RNAs (ncRNAs). Epigenetic control of gene expression is necessary and essential for typical organism development and cell control [63]. Epigenetic changes are transmissible and reversible changes that do not change the nucleotide sequence but cause changes in gene expression [64]. Changes that occur within the gene itself cause structural changes in some synthesized proteins. These changes lead up to early onset-OA. Given the above considerations, epigenetics is a very important area in the diagnosis, prognosis and treatment of OA [63]. Three different epigenetic regulation are involved in the molecular pathogenesis of OA. These include DNA methylation, expression of noncoding RNAs [ncRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs)], histone modifications that regulate gene expression at transcriptional and/or post-transcriptional levels [65]. DNA methylation is the most studied epigenetic control mechanism. 5-methylcytosine is formed as a result of the addition of a methyl group to the 5′ position of cytosine in the CpG dinucleotide by DNA methyltransferase [DNA Mtase (DNMT)]. Methylation at gene promoter regions is associated with suppression of gene expression. On the other hand, methylation within the gene bodies is associated with increased gene expression [66, 67]. The candidate gene study conducted to examine DNA methylation of matrix-degrading proteases such as MMP3, MMP9, MMP13 and ADAMTS4 was the first study to describe the possible effect of DNA methylation in OA. In the said study, hypomethylation was demonstrated in the promoter regions of selected catabolic genes in OA chondrocytes, and it was found that this hypomethylation was associated with increased expression of the gene [68].

miRNAs are small ncRNAs, which consist of 19 to 25 nucleotides and function at the post-transcriptional level by binding and repressing the expression of specific mRNA targets. miRNAs are involved in different cellular pathways and play a role in OA and in maintaining cartilage homeostasis [69]. Despite the constantly increasing number of publications and miRs related to the pathogenesis of OA, there is still no miR biomarker, which has been validated for use in the early diagnosis of the disease. This has been atrributed in part to the fact that OA is a multifactorial heterogeneous disease [63].

#### *Rheumatoid Arthritis*

lncRNAs are large RNA molecules comprising more than 200 nucleotides. Deregulated expression of lncRNAs plays an important role in inflammatory diseases. lncRNAs have been shown to be associated with OA progression and cartilage degeneration [70]. LncRNAs regulate gene expression at the post-transcriptional level via micro-RNAs and modulate transcriptional gene silencing through chromatin regulation [71].

#### **2.5 New treatments and future in osteoarthritis**

Since chronic low-severity inflammation is involved in OA, the development of drugs that act on pro-inflammatory cytokines has also become a new hope in the treatment of OA [72]. In a study, an intra-articular (IA) IL-1 receptor antagonist (IL-1Ra) was applied to the canine knee and it was reported that it reduced the number and size of osteophytes in the femoral condyle in the follow-ups [73]. However, in another study, anakinra, which is IL-1Ra, was applied IA to the knees of patients with OA. In this randomized controlled trial, they found no superior effect to placebo on pain and WOMAC scores [74].

It has been reported that the serum TNF levels of patients with OA are elevated. The positive results obtained with the use of TNF-α inhibitors especially in erosive hand osteoarthritis are promising. In the study of Magnano et al., 12 patients with erosive hand OA were treated with adalimumab (ADA) and reported a significant improvement in symptoms after 3 months [75].

Proteases are mediators that play a primary role in the catabolic process of OA. 'Agreccan', the largest proteoglycan, provides cartilage elasticity. The ADAMTS family of enzymes, also called aggrecanases (ADAMTS-4-5), is involved in the early stage of OA degeneration and is responsible for aggrecan degradation [28]. Preclinical studies of the molecule GSK2394002, which effectively inhibits ADAMTS 4 and 5, were discontinued because serious cardiovascular side effects were encountered in animal experiments with systemic use [76]. However, phase II studies on 114810, an IA administration molecule developed to reduce systemic side effects, are ongoing [77].

OA is a dynamic process triggered by various biochemical and mechanical factors, where destruction and repair are together. The fibroblast growth factor 3 (FGF-3) family, especially FGF 18, has an anabolic effect on human chondrocytes [78]. In a study including 549 patients with stage 2 and 3 knee OA, FGF-18 (sprifermin) IA was administered. An increase in tibiofemoral joint cartilage thickness has been reported up to 12 months. In the light of this information, it can be said that Sprifermin is currently one of the promising candidates for disease-modifying OA drug (DMOAD) [79].

The mechanism of action of platelet-rich plasma (PRP) is suggested to be that bioactive growth factors released from α granules in platelets stimulate tissue healing at high concentrations. In a meta-analysis of 16 studies, 1543 patients were examined; PRP and IA hyaluronic acid (HA) were compared. In terms of pain and functionality, it was found to be more effective than intra-articular HA injection [80]. However, the 2019 OARSI guidelines state that there is low-level evidence of the use of PRP in patients with knee, hip, and polyarticular OA and should not be used [81]. Larger randomized controlled studies with long-term follow-up are needed to elucidate its effects on tissue regeneration and delaying surgery.

In the light of these, the aim of OA treatment is to prevent disease formation or to provide regeneration of damaged tissue rather than eliminating the symptom. It would be more logical for DMOADs to be developed in the future to target the early stages of disease pathogenesis. For this reason, randomized double-blind controlled studies will contribute to the development of OA treatment.

*Pathogenesis, Pathology and Genetics of Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.99238*

#### **Author details**

Ferhat Ege Algology Department, Hatay Training and Research Hospital, Hatay, Turkey

\*Address all correspondence to: dr.ege\_ferhat@hotmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Pathogenesis, Pathology and Genetics of Osteoarthritis DOI: http://dx.doi.org/10.5772/intechopen.99238*

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#### **Chapter 2**

## Pathogenic Role of microRNA in Rheumatoid Arthritis

*JiuJie Yang, Jerome P.L. Ng, Kaixi Zhang, Liang Liu and Vincent Kam Wai Wong*

#### **Abstract**

Rheumatoid arthritis (RA) being a chronic inflammatory disease can be affected by both genetic and environmental factors. Abnormal functioning of immune response is the main underlying cause of RA. A growing number of studies on related diseases uncovered that microRNA (miRNA) may influence the pathogenesis of RA, such as the promotion of proliferation of fibroblast-like synoviocytes and secretion of cytokines by highly expressed miRNAs. A large number of studies have reported the aberrant expressions of miRNAs during the entire phase of RA, from the preclinical to terminal stages. These dynamic changes can be potentially developed as a bio-marker for predicting the risk, diagnosis and clinical management of RA. This chapter aims to summarize and discuss miRNAs' roles and mechanisms in the process of RA development, differential diagnosis from other diseases, clinical management and refractory RA. Therefore, miRNA demonstrates future perspectives of diagnosis and treatment of clinical RA under the support of newly discovered theoretical basis.

**Keywords:** Rheumatoid arthritis, microRNA, bio-marker, diagnosis, refractory rheumatoid arthritis

#### **1. Introduction**

Rheumatoid arthritis (RA) is an autoimmune disease, which causes joint deformity and disability in patients. RA can occur at any age, particularly with a high incidence in women aged 30–50 [1]. It has been shown that the average lifespan of RA patients is 3 to 18 years shorter than that of healthy people [2]. Patients with RA have high mortality rate and extra-articular complications, such as cardiovascular diseases, becoming the greatest challenge [2]. Proliferation of synovial tissue, infiltration of inflammatory factors, imbalance of immunity system, and destructions of bone and cartilage are the main common pathological characteristics of RA [3]. However, the current understanding of RA etiology and pathology are far yet to be elucidated. Some opinions on RA etiology illustrated high risk factors including but not limited to gene background, gender difference, smoking, obesity and environment factors. During the last decade, a growing number of evidence has shown that the epigenetic mechanism of microRNA (miRNA) regulation contributes remarkably to RA pathogenesis.

MiRNAs belong to the non-coding RNA family, with about 22 nucleotides in length. The processes of miRNAs biogenesis and maturation take place in the nucleus. The transcription of primary miRNAs (pri-miRNAs) from DNA molecule is the first step. After the recognition of these pri-miRNAs by an enzyme-protein complex, they are cleaved into precursor miRNAs (pre-miRNAs) with 70–100 nucleotides in length. Subsequently, the pre-miRNAs mature in the cytoplasm. Mature miRNAs finally regulate the post-transcriptional gene expression by binding to 3′-untranstaled region (3'UTR) of target mRNAs. Interestingly, the same gene can be modulated by multiple miRNAs, which collectively fine tune the expression of a certain gene. One-third of human genes of note is regulated by miRNA [4]. In addition, miRNAs participate the regulation of cell bio-behaviors, such as apoptosis, proliferation and invasion. Cytokine signaling is commonly known to regulate immune system, which is associated with the pathogenesis of RA. A large number of evidence showed that miRNAs participate in regulations of both innate and adaptive immunities by modulating cytokine signaling [5], such as the upregulations of miR-146 and miR-155 in LPSmediated innate immune response. Besides, a high expression level of miR-155 during thymic differentiation can increase Treg sensitivity to IL-2 and growth factors [6]. Given their important roles in cell regulation mechanisms and immunity responses, miRNAs have been frequently studied as potential bio-markers in diagnosis, target treatment, activity monitoring and therapy for RA disease. For example, during the early stages of undifferentiated arthritis, a high expression level of miR-483 was only found in patients who finally developed RA. In this chapter, we aim to review the different roles of miRNAs in RA, from the pathogenesis to clinical impact.

#### **2. The functions of miRNA in RA development**

Studies showed that synovial hyperplasia is a main pathological feature of RA, but the pathogenesis of RA is not fully elucidated. Fibroblast-like synoviocyte (FLS) is a major cell type found in the structure of synovial intima [7]. The most important step in the development of RA is the transformation of FLS by over-activation to RAFLS [8]. This process makes RA to present a characteristic, aggressive, and active clinical phenotype. It has been reported that RAFLS can recruit inflammatory cells through autocrine and paracrine methods to maintain the inflammatory state [7]. At the same time, compared with FLS, RAFLS has the characteristics of anti-apoptosis, predominant cell proliferation, invasion and metastasis. It also secretes inflammatory factors and promotes erosion of bone matrix (e.g. matrix metalloproteinases, MMPs). These secreted cytokines form a complex network system that affects each other, leading to an imbalance between synovial cell proliferation and apoptosis. This process therefore plays an important role in the progression of RA disease. Controlling the local proliferation of synovial cells and inducing their apoptosis are the key towards improvement of RA prognosis. Recent results showed that the activated phenotype of RAFLS is underpinned by epigenetic mechanisms—DNA methylation, histone modifications, and miRNA activity [9]. Newly emerging evidence suggested that dysregulated miRNA expressions in RA synovial tissues, especially in RAFLS, may generally contribute to the molecular mechanism of disease. Comparing miRNAs expression in FLS between RA and osteoarthritis (OA) patients, miR-124a was only down-regulated in RAFLS [10]. Further experiments revealed that overexpression of miR-124a can to suppress RAFLS proliferation. In contrast to miR-124a, miR-203 was up-regulated in RAFLS compared with healthy FLS [11, 12]. Importantly, a high level of miR-203 can target NF-кB signaling pathway, promote IL-6 and MMPs secretions, and support RAFLS invasion and migration [12]. Besides, there are lots of miRNAs like miR-126 [13], miR-152 [14], miR-137 [15], miR-199a-3p [16] and miR-338-5p [17], controlling the development of RA via regulating RAFLS.

*Pathogenic Role of microRNA in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99212*

RA is a well-known autoimmune disease, and both innate and adaptive immunities are the crucial steps for RA development. The role of miRNAs in regulating immune response has been reported in the literature. Alternations of miRNAs level can control the differentiation and immunological functions of various immune cells (monocytes, macrophages, and T cells) [18]. Many changes of miRNAs in these cells in RA patients have been reported. Chronically activated T cells are considered to be the trigger and key to RA. They are also the crucial link in inducing and aggravating RA immunological inflammatory response. On the one hand, they can induce activation of synovial macrophages and RAFLS. On the other hand, they contribute to T-Treg imbalance, which is a predominant mechanism of RA. A great number of studies have confirmed that there are various miRNA expressions modulating T cells, such as miR-17 [19] and miR-146a [20]. Additionally, RA patients showed the increases of miR-16, miR-103a, and miR-222 in peripheral blood mononuclear cells (PBMCs) of RA patients, which promoted cytokine secretion and disturb T-Treg balance [20]. The main miRNAs changes in different cells of patients compared with healthy controls were summarized in **Table 1**.


#### **Table 1.**

*Changes in miRNA level in RA patients compared to healthy individuals.*

Having a clear understanding of miRNAs in the regulation of RA pathogenesis provides a new direction and strategy for RA treatment. In some animal models, miRNA mimics or silencers were used to regulate miRNAs expressions, thereby reversing the inflammatory reaction or joint damage. One example is the amelioration of arthritis severity by reducing RAFLS's population via intra-articular injections of miR-124 and miR-140 mimics [32, 33]. Furthermore, intra-peritoneal injection of miR-223 silencer showed the same effect on relieving arthritis severity [34]. In a CIA mice model, intravenous administrations of miR-146a [35] and miR-708-5p [36] mimics were beneficial to prevent synovial hyperplasia and structural joint damage. Taken together, further investigations on the role of miRNAs in the pathogenesis of RA are of utmost importance for the treatment and delaying progression of RA, as well as developing novel targeted drugs.

#### **3. MiRNA as a potential bio-marker in RA diagnosis**

RA often begins insidiously with chronic developments of signs and symptoms, which may vary in intensity over many weeks. For most patients with new-onset RA, there is no obvious symptom in the early stage. Most of them show joint discomfort, which is difficult to distinguish RA from other diseases. In clinical practice, using rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibodies as diagnostic indicators are not sufficient [37]. Notably, the sensitivity and specificity of CCP antibodies in RA diagnosis were ~72% and ~92% respectively [38]. In some special cases, the CCP antibodies' titers cannot reach the diagnostic thresholds. Moreover, genetic and environmental risk factors, together with systemic immunization, affect the multi-stage development of RA. Identifying patients with RA and providing them a proper treatment can prevent 90% of patients in early-stage period from the progression of joint damage, and improve prognosis [39]. Therefore, there is an urgent need for identifying novel bio-markers to screen high-risk individuals and those with early stage of RA.

Single nucleotide polymorphism (SNP) variants residing within boundaries of genes encoding miRNAs is a common phenomenon, which may affect multiple major human disorders including RA [40]. The associations between miRNA-linked SNP and RA susceptibility have been studied extensively, such as rs11761231 in miR612, rs615672 in miR-541, rs2837960 in miR-509/602, rs6684865 in miR-181, rs9550642 in miR-1238 and rs6920220 in miR-519 [41]. Furthermore, the association of variations of miRNA target genes with RA was exemplified by the discovery of SNP rs3027898 variant in miR-146a target gene, IL-1 receptorassociated kinase (IRAK-1), in RA patients. Collectively, the alterations of miRNA gene and its target gene may increase the risk of developing RA.

The current understanding of the role of miRNAs in RA pathogenesis is limited, especially in the preclinical phase of RA. Some serum miRNA expression profiles from different people were evaluated to determine mechanisms underpinning the progression of RA onset in at-risk individuals. Among those miRNA expressions, only miR-103a-3p specifically increased in both RA patients and their seropositive first-degree relatives [42]. Patients who have symptoms of non-differential arthritis and a high serum miR-22 expression, finally developed RA [20]. Recently, a study examined circulatory miRNAs in RA patients, and further investigations illustrated that miR-221-3p, let-7d-5p, miR-431-3p, miR-130a-3p, miR-126-3p and miR-24-3p were significantly elevated in subjects "at risk" of developing RA [42]. Particularly, the elevated whole blood level of miR-103a-3p may become a powerful bio-marker for positive anti-citrullinated peptide antibodies (ACPA) individuals who have possibility to develop RA [42].

Early stage RA (ERA) is defined as a disease duration less than 12 months. Several clinical studies have shown that ERA is a "window of opportunity" for disease-modifying anti-rheumatic drug (DMARD) therapy. Most patients at this stage will get long-term remissions or even complete remissions after systematic treatments. There are no golden diagnostic criteria in ERA to date, although this stage is important in clinical practice. During the last decade, multiple studies have demonstrated miRNA as a powerful tool for identifying molecular bio-markers for diagnosis in ERA. One highlight example is the analysis of highly expressed miR-22 level for distinguishing ERA patients from healthy individuals. Besides, miR-16, miR-146a, miR-223 and miRNA-155 were significantly down-regulated in ERA, and even lowered in established RA and healthy controls [43–45]. Generally, these miRNAs possibly improve early diagnosis of RA, especially in sero-negative patients.

RA diagnosis not only distinguishes the different phases of RA, but also differentiates RA from other diseases, such as systemic lupus erythematousus (SLE), OA, multiple sclerosis (MS). Those diseases show similar symptoms to RA at the beginning. Several studies have established analyses of different expressions of miRNAs among those indistinguishable diseases. Compared with healthy people, miR-146a and miR-155 were up-regulated in PBMCs of RA cases, and conversely, they had low expressions in PBMCs of SLE patients. In addition, miRNA-516a-3p, miRNA-629 and miRNA-525-5p levels in PBMCs were significantly up-regulated in active SLE patients compared with those in healthy controls [46], but all these miRNAs have no specific expressions in RA patients. A recent study revealed that the expressions of miR-371b-5p and miR-5100 also increased notably in the serum of SLE compared with healthy control and RA [47]. Further results revealed that miRNA-346 in synovial tissues was only specifically elevated in RA [48]. Another seven miRNAs expressions in macrophages from patients with active RA and OA were also recently determined. MiR-99a, miR-100, miR-125b, miR-199-3p, miR-199-5p, miR-152 and miR-214 were down-regulated in macrophages in RA, while only miR-223 was up-regulated, compared with OA samples [49]. One more example is that the expression level of miR-34a-3p in RAFLS was generally lower than that in OAFLS [50].

Clearly, the observable changes in miRNAs and their molecular networks are of great values for determining new mechanisms related to the onset of RA, and also being used as bio-markers to predict the onset of preclinical RA and distinguish RA from other diseases.

#### **4. The application values of miRNAs in RA clinical management**

#### **4.1 MiRNAs' functions in activity monitoring of RA**

The clinical management strategy of RA is "treat-to-target" [51]. In other words, patients can achieve remission or at least low disease activity state within 6 months after effective treatment. If RA is insufficiently treated, extra-articular manifestations, such as the most frequently occurring rheumatoid nodules and even cardiovascular disease, may occur. Notably, this kind of cardiovascular disease is more closely associated with RA disease activity rather than traditional cardiovascular risk factors. Furthermore, either manifestation of RA or complication of RA therapies (e.g. MTX and leflunomide) may lead to interstitial lung disease (ILD). This affirms the importance of activity monitoring from different aspects. Hence, it is necessary to develop new treatment strategies to retard RA progression by quick identification of conditions of RA remission before irreversible damage in joint

[39]. Currently, clinical indicators for RA activity monitoring are mainly based on clinical, laboratory and physical examinations, including simplified disease activity index (SDAI), disease activity score 28 (DAS28), erythrocyte sedimentation rate (ESR), C reaction protein (CRP) [52]. These indicators can be affected by subjective and objective factors, such as OA, fibromyalgia, and assessor's experience. Both ESR and CRP are non-specific markers of inflammation, which are commonly affected by age, anemia, immunoglobulin and other factors. Therefore, these markers are not specific enough to RA patients. In view of the clinical demands, it is particularly important to develop effective, precise and accurate biological markers for the evaluation of RA disease activity. Recent studies demonstrated that miRNA, a potential bio-marker, can be aberrantly expressed in different stages of RA progression, and thus allowing to monitor disease activity.

The correlations of miRNA levels (miR-125b, miR-21, miR-155, miR-346, miR-223 and miR-146a) in PBMC of RA patients with clinical characteristics and inflammation markers in RA patients were reported [53]. The expression levels of miR-146a and miR-155 were positively related to ESR, DAS28-CPR and cytokines (IL-1β, IL-17α, IL-6 and TNF-α). On the contrary, miR-21 was negatively related to DAS28 and those cytokines. Another study found that miR-125b was inversely correlated with RA activity [54]. The studies on miR-24 in patients' serum with active RA disease uncovered that the miR-24 level increased with ESR and the DAS28 [55]. Besides, miR-5571-3p and miR-135b-5p levels were found to be positively correlated with the disease activity and the inflammation level of RA [56]. Notably, the upregulated expressions of hsa-miR-432-5p and especially hsa-miR-194-5p in serum were associated with relapse in RA patients [57]. Increasing serum level of miR-223 was also found in remission patients several days before RA relapse [20]. Moreover, blood samples from 76 RA patients illustrated that lowering the levels of miR-548a-3p can promote RA relapse or increase disease activity [31]. In some cases, RA patients without proper treatment were accompanied by extra-articular symptoms, together with changes in some miRNAs levels. Analyzing abnormal expressions of miRNAs can assist in diagnosis of RA-related diseases. For example, some researchers collected miRNAs (e.g. let-7c-5p, miR-30a-5p, miR-30e-5p, miR-125a-5p, miR-126-3p, miR-126-5p, miR-425-5p, miR-3168, and miR-4446-3p) in a panel to predict cardiovascular disease in patients with RA [58]. Other findings found that differences in circulating miR-200c levels can distinguish RA patients with and without ILD [59]. More examples of the relationship between miRNAs expression and RA activity were shown in **Table 2**.

#### **4.2 MiRNAs as potential bio-markers of therapeutic effectiveness**

Despite the great progress of management of RA over the past three decades, anti-rheumatoid drugs (DMARDs), including conventional synthetic DMARDs (csDMARDs) and specific targeted DMARDs (e.g. biologic DMARDs, b-DMARDs, and targeted synthetic DMARDs, tsDMARDs), are still the first-line drugs for RA patients; however, a certain number of patients does not benefit from the treatments with multiple DMARDs [65]. For those patients, biological treatments targeting inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukins (ILs), and B or T cells may give a better outcome [66–69]. Nevertheless, ~20–30% of patients fail to respond to these biological agents. Therefore, it is necessary to explore novel bio-markers for predicting the clinical responses of RA patients to DMARDs or other therapies. The current indicators for assessing the therapeutic effectiveness involve inflammatory factors, disease activity and patient response outcome (PRO) [70]. These evaluation indicators, however, are easily affected by many factors, resulting in certain deviations. Emerging research showed

#### *Pathogenic Role of microRNA in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99212*


#### **Table 2.**

*The relationship between miRNAs expression and RA activity.*

that the prediction of RA treatment was possibly achieved by monitoring the alterations of miRNA levels. This encourages the development of novel therapeutic strategies for RA via identifying molecular mechanisms of miRNAs.

Several findings demonstrated that almost all miRNAs expressions were changed during therapy. Using MTX significantly decreased the expressions of miR-155 and miR-146a, but increased the expression of miR-34 in rat tibiotarsal tissues [71]. Clinical research proved that RA patients who responded to MTX had lower expressions of specific miRNAs, including not only hsa-miR-155-5p and hsa-miR-146a-5p, but also the newly reported hsa-miR-132-3p [60]. The circulating miR-10a in RA patients was markedly decreased, but was up-regulated when treated with MTX [72].

Although miR-223 and miR-16 were shown to be overexpressed in synovial tissues of RA patients, their expressions were decreased after treated with csDMARDs [62]. More importantly, disease severity was reduced when miR-223 was silenced in experimental arthritis [34]. Another finding demonstrated that miR-125b expression showed more alternations between patients in terms of good response and poor response [73]. Its expression was relatively low in patients with early RA, but increased markedly after 3 months of conventional therapy [54]. Thus, these miRNAs could become potential bio-markers in both csDMARD and bDMARD therapies.

Furthermore, some miRNAs were good candidates for predicting the treatment of RA with anti-TNF therapy. A placebo-controlled, double-blind and prospective study of patients with early RA showed that the highly expressed miR-886.3p in combination with lowly expressed miR-22 were associated with the probability of EULAR good response (~95%) [74]. This may indicate the responses of miR-22 and miR-886.3p to adalimumab treatment in RA. RA patients before TNF-α therapy showed a relatively higher miRNA-5196 expression than those treated with anti-TNF-α therapy and healthy controls [75]. Studies implied that an increase of miR-155 may result in the upregulation of membrane TNF expression on monocytes and the defect of monocyte capacity to differentiate into M2-like anti-inflammatory

#### *Rheumatoid Arthritis*

macrophages, which were the clinical characteristics specific to RA. Notably, increased miR-155 could be partially reversed by monoclonal anti-TNF antibodies [76]. Besides, the expressions of miR-126, miR-148a, miR-29c, miR-30c, miR-17, miR-21, miR-223 and let-7b in neutrophil of RA patients were declined after treatment with anti-TNF-α drugs [77]. Obviously, these miRNAs could be the potential bio-markers for DMARDs therapy in RA.

Taken together, dynamic changes of miRNAs were not only associated with disease activity, but were also affected by therapeutic effects. This reflects the potential clinical values of miRNAs expression as novel prognostic markers for RA patients, in terms of RA management.

#### **5. MiRNA in refractory rheumatoid arthritis**

Most patients achieve remission or low disease activity state with effective therapies and treatment strategies. However, about 20–25% of the patients do not reach a state of low disease activity, and the causes of refractory rheumatoid arthritis (RRA) have not been identified. The RRA may attribute to the epigenetic changes accumulated by chronic RA. A large amount of evidence indicated that changes in miRNAs can occur either before or after treatment. Therefore, the alterations of miRNAs may affect the duration of RA or the therapeutic effect, leading to RRA. However, the mechanisms of miRNAs mediating RRA are still largely unknown. Up to date, the mechanism of miRNA on RRA has been known to be related to the regulation of drug efflux transporters, apoptosis and cell cycle modification. Notably, some somatic genes, such as p53, may also influence RA via miRNAs.

ATP-binding cassette (ABC) transporters are located in cell membrane responsible for transporting endogenous metabolites and xenobiotics across cell membranes in an ATP-dependent manner [78]. The high expression levels of ABC transporters were commonly found in cells from the inflammatory area of refractory RA patients [79]. These abnormal expressions in RA patients subsequently increased drug efflux and caused patients a lower response to treatment. The reduction of therapeutic effect of MTX by increased expression of ABCB1 in RA patients is a distinguishable example [80]. Importantly, the MTX-treated group showed the ABCC1 expression in synovial tissues higher than the untreated group [81]. These studies corroborated that the increasing MTX resistance in RA patients may result from the upregulations of ABC transporters. Hence, declining ABC transporter expression provides a potential solution for reversing drug resistance in RA chemotherapy. One of the reasons for miRNAs being as potential therapeutic targets for chemoresistant cancers is their regulations on the expression of ABC transporter. Research in ovarian cancer demonstrated that miR-522 inhibited ABCB5 in HT29 colon cancer cells to reverse drug resistance to doxorubicin [82]. ABCG2-mediated drug resistance to 5-FU in colon cancer side population cells was overcome by overexpressed miR-34a via suppressing DLL1 expression [83]. Similarly, ABCB1 (P-gp) expression was downregulated by miR-491-3p via directly bound to the 3'-UTR of ABCB1 [84]. MiR-214-3p also acts as a tumor suppressor to inhibit chemoresistance in retinoblastoma by targeting ABCB1 [85]. MiR-1268a regulated ABCC1-mediated drug resistance to temozolomide in glioblastoma [86].

Based on the important role of RAFLS in RA development, most drugs achieve the remission of RA by controlling RAFLS-related activities. MiRNA has been considered as a potential reason for refractory RA owing to its important role in regulating RAFLS mechanisms. On the one side, miRNA promoted the secretion of pro-inflammatory cytokines or MMPs; and increased RAFLS proliferation, invasiveness, survival and anti-apoptosis. On the other side, they can regulate various

#### *Pathogenic Role of microRNA in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99212*

intracellular pathways in RAFLS, which commonly include Wnt, NF-κB, JAK/STAT and TLRs signaling pathways. For example, reduced miR-20a expression in RASFs activated the JAK-STAT3-mediated inflammation, and promoted cell proliferation and apoptosis-resistance [87]. The regulation of PI3K/AKT pathway by targeting PIK3R2 with miR-126 promoted RA synovial fibroblasts proliferation and apoptosis-resistance [88]. In a separate study, miR-650 was down-regulated in RA patients compared with patients with joint trauma undergoing joint replacement surgery [89]. Further study confirmed that miR-650 targeted AKT2 to promote FLS proliferation and migration, and reduce apoptosis. In another example, down-regulated miR-375 in an AIA rat model aggravated the inflammation of FLS through Wnt signal pathway [90]. Interestingly, the expression level of the same miRNA varied in different tissues, along with different functions. One example is miR-21, which increased significantly in a rat model of collagen-induced RA with the promotion of FLS proliferation via NF-kB pathway [91]. In contrast, the miR-21 level in RA patients was declined due to the participation in the imbalance of Th17 and Treg cells [92].

Generally, p53 being as a tumor suppressor regulates many signaling pathways like apoptosis, cell cycle, DNA repair, and cellular stress responses by modulating the expressions of miRNAs [93]. Since p53 plays important roles in inflammation, apoptosis, and cell proliferation, the p53 function lost by gain-of-function (GOF) mutation or its low expression influences RA pathogenesis. Similarly, GOF mutation of p53 can confer tumor cell oncogenic properties such as chemoresistance and invasion. According to statistical investigations, the mutation rate of p53 gene in RA patients was about 50% [94]. In particular, a pro-apoptotic molecule, p53-regulated apoptosis-inducing protein 1 (p53AIP1) was suppressed by p53 mutation (from arginine to glutamine at site 248) in RAFLS, leading to an anti-apoptotic effect [95]. However, the mechanisms of p53-mediated apoptosis resistance are yet to elucidate.

Typically, wild-type p53 regulates miRNAs to work. For instance, p53 controlled cell apoptosis through regulating miRNAs expressions (e.g. miR-34a, miR15a, and miR16–1) [93]. In RA patients, miR-15a and miR16–1 initiated anti-apoptosis by inhibiting anti-apoptotic molecule B cell lymphoma 2 (Bcl2) [96]. In addition, miR-34a expression in RA-FLSs was positively related to X-linked inhibitor of apoptosis protein (XIAP) expression which induced RAFLS anti-apoptosis [97]. Since p53 activates all the above-mentioned miRNAs, functionally defective p53 (p53 mutation) may influence RAFLS apoptosis resistance.

Cyr61, which is a secreted and cysteine-rich extracellular matrix (ECM) protein produced by RAFLS, is stimulated by IL-17 for FLS proliferation [98]. Over-expressed Cyr61 is an important mediator in a malicious cycle, where a high level of Cyr61 promotes RAFLS proliferation and Th17 cell differentiation [99]. Generally, wild-type p53 from RA patients promoted expression of miR-22 targeting the 3-UTR of Cyr61, leading to a decrease of Cyr61 expression [100]. However, functional defect of mut-p53 in RA synovial tissue was unable to activate miR-22 expression, causing abnormally high Cyr61 expression and, in turn, promoted RAFLS proliferation and IL-6 production [100]. Thus, a reduced miR-22 level in RA synovial tissue and the resulting RRA attributes to somatic mutations of p53.

MiR-155 is also an important regulator in the pathogenesis of RA. Highly expressed miR-155 in PBMCs of RA patients was positively related to inflammatory cytokine (e.g. TNF-α and IL-1β), RA activity laboratory indicators (CRP, ESR) levels and DAS28 respectively [101]. Recent study indicated that mut-p53 increased miR-155 expression in breast cancer, which accelerated cell proliferation, epithelialmesenchymal-transition (EMT) and invasion [102]. This implied that p53 mutations may affect the process of RA via miR-155 as similar to breast cancer.

Overall, miRNAs are not only an independent factor that affects the refractory of RA, but also are an intermediate link of certain gene mutations related to RRA. This may provide a new direction for treating refractory RA or reversing miRNA-related apoptosis resistance.

#### **6. Conclusions**

MiRNA, a non-coding RNA, widely exists in tissue cells and body fluids. It is worth mentioning that the studies on miRNA in RA are still limited, but the results verify its important role in immune response regulation and sustained inflammatory response to date. SNPs in specific miRNA genes, such as miR-541, are related to the high risk of RA development. Moreover, most miRNAs in synovial tissues can influence the process of RA by regulating RAFLS proliferation, invasion and apoptosis via targeting inflammatory or immune signaling pathways like NF-κB and Wnt pathways. Current efforts have confirmed that the expression level and mechanism of the same miRNA varies in different tissues or cells from RA. For example, miR-21 level in PBMCs was declined to regulate Th-Treg balance by targeting STAT3, STAT5 and Foxp3, but that in RAFLS was overexpressed to promote proliferation of RAFLS through NF-κB signaling pathway. For clinical management, the dynamic change of miRNAs can be a bio-marker for monitoring disease activity and therapeutic response, as exemplified by the association of high miR-223 level with high disease activity and RA relapse. In addition, some miRNAs may play a crucial role in regulating refractory RA or drug-resistance RA.

Finally, an increasing extent of data demonstrates the importance of miRNAs to the regulation of the RA process, along with its potential developments in biomarker discovery and special targets for treatment. Hence, understanding miRNA analysis can be served as a diagnostic and/or prognostic tool in an array of inflammatory disorders, especially RA.

#### **Acknowledgements**

This work was supported by a FDCT grant from the Macao Science and Technology Development Fund (Project code:0003/2019/AKP) and Foshan Medicine Dengfeng Project of China (2019-2021).

#### **Conflict of interest**

The authors declare no conflict of interest.

*Pathogenic Role of microRNA in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99212*

#### **Author details**

JiuJie Yang1,2, Jerome P.L. Ng1 , Kaixi Zhang1 , Liang Liu1 \* and Vincent Kam Wai Wong1 \*

1 Dr. Neher's Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China

2 Macau Medical Science and Technology Research Association, Macau, China

\*Address all correspondence to: lliu@must.edu.mo and bowaiwong@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Role of LncRNA in Rheumatoid Arthritis

*Ayse Kocak*

#### **Abstract**

Long non-coding RNAs (lncRNAs) are a class of non-coding RNA (ncRNA) molecules that do not have protein coding. They are ubiquitous in the process of transcription and gene regulation. lncRNAs regulation is correlated with many diseases. Rheumatoid arthritis (RA) is a chronic inflammatory disorder and this disease can affect especially joints. Nevertheless, in some patients, RA and inflammation can damage body parts such as the eyes, lungs, skin, heart, and blood vessels. Lots of lncRNAs were confirmed to be correlated with rheumatoid arthritis (RA) pathogenesis. Particularly GAPLINC, ZFAS1, PTGS2, and HOTAIR lncRNAs play a role in RA. This chapter will be explained and summarized the relationship between IncRNAs and RA.

**Keywords:** lncRNA, rheumatoid arthritis

#### **1. Introduction**

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. It is associated with progressive joint destruction complications and decreased life expectancy [1]. RA's main clinical features are typically symmetrical polyarthritis with swelling, redness, and pain in the distal joint, particularly the small joints of the hands and feet [2]. Advances in understanding the pathogenesis of the disease, RA treatment greatly improved with an emphasis in the early stage. To our best knowledge, lots of laboratory tests used for RA generally include rheumatoid factor (RF), c-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and anti-cyclic peptide containing citrulline (anti-CCP) antibodies [3]. Nevertheless, RA pathogenesis is still unclear, but it is most likely related to the physiological structure of the joint and anatomy [4].

Long non-coding RNAs' (lncRNAs) lengths are greater than >200 nucleotides, which are subclassified into five categories that include the natural antisense lncRNAs according to their positions relative to protein-coding genes, long intergenic ncRNAs (lincRNAs), intronic lncRNAs, bidirectional lncRNAs, sense-overlapping lncRNAs [5, 6]. Referring to the GENCODE database (version 31), 17,904 lncRNA genes were identified in the human genome. lncRNAs play a role as critical regulators of cellular processes and disease stage or progression. lncRNAs have been studied in cancer [7, 8], innate and adaptive immunity [9], and inflammation [10]. Recently, studies of the role of lncRNA in RA pathogenesis are increased. Sequencing or microarray analyses revealed the expression profiles of lncRNAs in RA. The lncRNA expression profile in RA is different in various immune cell types such as B cells, natural killer (NK) cells, and T cells, indicating immune cell-type specificity of lncRNA expression. Identification of aberrantly expressed lncRNAs in RA and investigation of the underlying molecular mechanisms.

Emerging evidence suggests that lncRNAs are involved in the development of RA. Although numerous aberrant-expressing lncRNAs (HOTAIR, MALAT1, GAPLINC, PVT-1, LERFS, GAS5, DILC, NEAT-1, Lnc-p21, THRIL, RMRP, NTT, MEG3, Lnc-IL7R, ZFAS1, UCA1, C5T1LncRNA) have been reported in RA [11, 12], only a few of them are functionally determined. In this chapter, we summarize here the current findings of lncRNAs that may be involved in the pathogenesis of RA, aiming to encourage future research on this topic.

#### **2. Long non-coding RNAs in RA**

#### **2.1 Long non-coding RNAs**

LncRNAs play epigenetic regulation, cell cycle regulation, and cell differentiation and genetic roles [13] such as physiological and pathological and regulator process; also, lncRNAs are Central regulators of the immune response, but they are poorly conserved in species [14]. LncRNAs regulate the coding genes directly various molecular mechanisms [15]. LncRNAs expressed differentially and effects on immune cells in autoimmune diseases. The regulatory mechanism of lncRNAs is complex and needs to be investigated by more functional and mechanistic experiments. A group of lncRNAs is associated with clinical indicators such as CRP, ESR, serum proinflammatory cytokines, and DAS28, suggesting that lncRNAs may serve as biomarkers to monitor RA activity.

#### *2.1.1 LncRNA HOTAIR*

In RA patients, the expression of HOX transcript antisense RNA (HOTAIR), HOTAIR is decreased in fibroblast-like synoviocytes (FLSs), also HOTAIR suppresses the activation of MMP-2 and MMP-13. lncRNA HOTAIR, miR-138, and NF-kB axis have also been established in chondrocytes in RA, LncRNA HOTAIR may target miR-138 and inhibit the activation of NF-kB pathway [16], and the expression of HOTAIR increases in peripheral blood mononuclear cell and blood exosomes using lncRNA array analysis [17].

#### *2.1.2 LncRNA MALAT1*

In RA FLSs, MALAT1 plays a role regulation of cell proliferation and inflammation [18]. MALAT1 binds to the beta-catenin promoter in the WNT signaling pathway [19]. One group study suggests that MALAT1 plays a role in apoptosis proteins [19]. MALAT1 silencing suppressed Bax, and Bcl-2, caspase-3, caspase-9 in RA FLSs [19].

#### *2.1.3 LncRNA GAPLINC*

LncRNA long intergenic non-coding RNA (GAPLINC) may play act as a molecular sponge of miR-382-5p and miR-575. There is a negative correlation observed between the expression of GAPLINC and the miRNAs [20]. Also, GAPLINC may be a new therapeutic target for RA [21].

#### *2.1.4 LncRNA PVT-1*

Knockdown of plasmacytoma variant translocation 1 (PVT-1) in RA's FLSs suppresses the TNF-α and IL-1β pro-inflammatory cytokines [22]. In the same study,

#### *Role of LncRNA in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99525*

PVT1 regulates inflammation and apoptosis in RA-FLSs through the Sirt6 demethylation. Furthermore, PVT-1 increase at synovial tissue of RA patients and RA model and PVT-1 bound to miR-543 positively regulated the expression of signal peptide-CUB-EGF-like containing protein 2 (SCUBE2) by inhibiting the miR-543, guide to FLSs inhibition of apoptosis and IL-1β secretion. Inhibition of PVT1 may be a new idea for the treatment of RA [23].

#### *2.1.5 LncRNA LERFS*

Lowly expressed in rheumatoid fibroblast-like synoviocytes (lncRNA LERFS) negatively regulated the invasion, proliferation, and migration of joint synovium by interacting with heterogeneous nuclear ribonucleoprotein Q (hnRNP Q ) but in RA FLSs, LERFS is low expressed in RA FLSs and the reduced LERFS led to the reduction of LERFS-hnRNP Q complex [24]. In this study, LERFS regulates the expression and activity of CDC42, Rac1, RhoA and, probably by binding to the hnRNP Q complex.

#### *2.1.6 LncRNA GAS5*

In RA FLSs, LncRNA growth arrest-specific transcript 5 (GAS5) overexpression organizes cell apoptosis by activating cleaved caspase-9 and caspase-3 and inhibits PI3K/AKT signaling pathway [25, 26]. Also, GAS5 plays a role in the inflammatory response in RA. In synovial tissue and FLSs, GAS5 expression is decreased and expression of homeodomain-interacting protein kinase 2 (HIPK2) increases significantly and GAS5 reduced the level of TNF-α and IL-6 [27]. Also, overexpression of LncRNA GAS5 affects IL-18 levels, and IL-18 is downregulated by LncRNA GAS5 [28].

#### *2.1.7 LncRNA DILC*

DILC of plasma RA patients was downregulated, while IL-6 was upregulated and DILC level is negatively correlated with RA. DILC overexpression promoted the inhibition of IL-6 expression and FLSs apoptosis and in RA [29].

#### *2.1.8 LncRNA NEAT-1*

NEAT-1 is significantly upregulated in th17 cells differentiated CD44 cells from RA patients. Also, upregulation of NEAT-1 plays a role differentiation of CD4+ T cells into Th17 cells by regulating its downstream molecule STAT3 [30].

#### *2.1.9 LncRNA Lnc-p21*

In RA, Lnc-p21 expression is so low and can be renovated by the methotrexate treatment [31]. This LncRNA-p21 suppresses inflammation and is downregulated [31].

#### *2.1.10 LncRNA THRIL*

According to the information obtained from RA, THRIL is on the upward path [32]. This LncRNA may use as a biomarker for RA. THRIL expression in the blood of RA patients was positively correlated with TNF-α and erythrocyte sedimentation rate. THRIL inhibition is reversed the regulatory effect of TNF-α, and significantly reduced the activity of p-AKT and phosphoinositide 3-kinase (PI3K) signaling

pathways. Also, expression of THRIL may promote the activating of the PI3K/AKT signaling pathway, and this leads to the result of inflammation and proliferation of FLSs [33, 34].

#### *2.1.11 LncRNA RMRP*

LncRNA RMRP expression is high in T cells from patients with RA [32]. Also, LncRNA RMRP has a positive correlation with RA progression [35]. This LncRNA may be a biomarker for RA.

#### *2.1.12 LncRNA NTT*

NTT expression is increased in a peripheral blood mononuclear cell (PBMC) from early patients with RA [36]. In the same study, the researchers found that in RA, lncRNA NTT/PBOV1 is capable of regulating monocyte differentiation.

#### *2.1.13 LncRNA MEG3*

The level of LncRNA maternally expressed gene 3 (MEG3) is significantly downregulated in FLSs of patients with RA [37]. Also, this study suggests that in lipopolysaccharide (LPS)-treated chondrocytes LncRNA MEG is downregulated. Overexpression of LncRNA MEG3 has an inhibitory effect on RA pathology can be achieved by increasing the rate of chondrocyte proliferation through negative regulation of miR-141 and AKT/mTOR signaling pathway [38–40]. In RA patients, low MEG3 expression correlated negatively with serum level of HIF-1α and vascular endothelial growth factor A (VEGF) and positively correlated with BAX. MEG3 gene rs941576(A/G) polymorphism has been confirmed to be associated with increased RA severity in the population [41]. We can say that LncRNA MEG3 promotes proliferation and it has an inhibitory effect.

#### *2.1.14 LncRNA Lnc-IL7R*

LncRNA long noncoding-interleukin-7 receptor (Lnc-IL7R) inhibits apoptosis and leads to proliferation [42]. Also, Lnc-IL7R interacts with the enhancer of zeste homolog 2 (EZH2) to assist the FLSs' growth and it is necessary for PRC2-mediated inhibition of the cyclin-dependent kinase inhibitors 1A and 2A [42].

#### *2.1.15 LncRNA ZFAS1*

Ye et al. found that LncRNA ZFAS1 has abnormal activity in RA FLSs. Also, the knockout of LncRNA ZFAS1 suppresses the migration and invasion of FLSs and it takes miR-27 s as a target and increased the expression of miR-27a [43].

#### *2.1.16 LncRNA UCA1*

LncRNA UCA1 expression is low in RA FLSs [44]. It reduces caspase-3 and cell apoptosis *via* Wnt-6 [44].

#### *2.1.17 C5T1LncRNA*

LncRNA C5T1LncRNA is a new LncRNA and it inhibits the mRNA of C5 protein, this protein plays a role in inflammation in RA [45, 46].

#### **3. LncRNA as novel RA biomarkers**

As with other pathological and chronic diseases, RA affects patients' life and status. Many pieces of evidence have confirmed the role of lncRNAs in the pathogenesis of RA [47]. Luo et al. found 5.045 irregular lncRNAs in PBMCs (2.635 downregulated and 2.410 upregulated) of RA patients compared to controls [48], 135 potential lncRNA-mRNA target pairs and RP11-498C9.15 targeted RA-related genes and pathways. Lots of LncRNAs such as PVT-1, MEG3, HOTAIR suggest that LncRNAs may serve as novel biomarkers to monitor RA pathogenesis.

### **4. Discussion**

LncRNAs are of great importance in gene regulation and various RA biological processes. Expression profiles of lncRNAs vary in PBMCs, serum exosomes, osteoclasts, FLS, synovial tissues, plasma, synovium in RA. Some of these are differentially expressed, and LncRNAs are related to RA activity.

### **5. Conclusion**

Emerging evidence shows us that lncRNAs are important regulators in RA. Continuing to explore the functions of lncRNAs in RA, their aberrant expression profile, and determining their role and mode of action will help us understand the underlying causes of the disease. Also, the identified lncRNAs related to the pathogenesis of RA may be potential diagnostic markers or target molecules that regulate RA progression. In the future, LncRNA-based therapeutic tools will likely lead to treatment insights into RA.

### **Author details**

Ayse Kocak Kutahya Health Sciences University, Kutahya, Turkey

\*Address all correspondence to: kocak.ayse@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 2
