MicroRNAs: emerging players in apical periodontitis

Abstract Apical periodontitis is an inflammatory disorder of periradicular tissues developed from endodontic infections. Understanding its pathophysiology and the underlying molecular mechanisms is key to the advancement of endodontics. MicroRNAs (miRNAs), a group of evolutionarily conserved small non-coding RNAs, may be phenotypically and functionally associated with the pathogenesis of apical periodontitis. Several studies have focused on the role of miRNAs in the pulp and periradicular biology, and they have demonstrated their essential functions, such as initiating odontogenic differentiation and promoting pro- or anti-inflammatory responses in pulpitis. Up to date, over 2,000 miRNAs have been discovered in humans; however, only few have been reported to associate with apical periodontitis. Therefore, identifying miRNAs involved in diseased apical tissues and conducting functional studies are important in expanding our current knowledge of pulp and periradicular biology and exploring novel therapeutic avenues. In this review, we revisit current models of apical periodontitis and miRNA biogenesis, analyze existing evidence of the involvement of miRNAs in diseased apical tissues, and discuss their diverse functions and potential values. Based on their sheer abundance, prolonged stability in biofluid, and relative ease of sampling, miRNAs may be a useful tool to be developed as diagnostic biomarkers for apical periodontitis. Furthermore, it can be used as therapeutic targets in conjunction with conventional endodontic therapies.


Introduction
Microbes in the oral environment are the primary cause of infection in the root canal system as well as dental pulp and periapical tissues. 1  LPS evokes host immune response by activating pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and CD14 signaling. These signaling events subsequently induce the production of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-8, IL-12, IL-17, and anti-inflammatory cytokines such as IL-4 and IL-10. [5][6][7] Periapical inflammatory responses can cause tissue damage and the development of granulomas or radicular cysts by a complex cellular and molecular cascade. 8 For instance, matrix metalloproteinases (MMPs) -a group of more than 25 secreted and membrane-bound enzymes -are regulated by proinflammatory cytokines and growth factors, and play one of the key roles in tissue destruction and normal tissue modeling. 9 In particular, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13 have been found in periapical lesions. Collagen, as well as extracellular matrix (ECM) degradation, have been associated with increased levels of MMPs. 9 In addition to pathophysiological changes in connective tissues, periapical inflammation also leads to alveolar bone resorption. 3 Bone turnover depends on the equilibrium between osteoblastmediated bone formation and osteoclast-mediated bone destruction, and the osteoclast activities are regulated by the receptor activator of NF-kappa B (RANK), its ligand RANKL, and the decoy receptor of RANKL, osteoprotegerin (OPG). 10 The binding of RANKL to RANK drives the maturation of osteoclasts.
Studies have shown that pro-inflammatory cytokines can induce the expression of RANKL, which leads to increased osteoclast activities and subsequent bone resorption. 10 It is noteworthy that complex interactions between inflammatory cells and cytokines influence the balance between pro-and anti-inflammatory processes that, in turn, affect the equilibrium between destruction and regeneration of apical tissues. 11 For example, anti-inflammatory cytokines, such as IL-4 and IL-10, can restrain inflammatory cytokine production and inhibit the function of MMPs and RANKL by stimulating the production of tissue inhibitors of matrix metalloproteinases (TIMPs) and OPG, respectively. 12 MicroRNA MicroRNAs (miRNAs) are a class of evolutionally conserved non-coding RNAs of ~23 nucleotides that regulate gene expression predominantly at the posttranscriptional level. 13 Since its initial discovery of lin-4 in Caenorhabditis elegans in 1993, hundreds of miRNAs have been identified and extensively studied in a wide spectrum of species. 14,15 Mammalian miRNAs are encoded in both intergenic and intragenic regions of the genome. 16 In general, miRNA genes are first transcribed into long primary transcripts, called pri-miRNAs. Pri-miRNAs are processed by the nuclear RNase III Drosha/DGCR8 to generate ∼70 base pair-long pre-miRNAs that are then exported by Exportin-5 to the cytoplasm in a Ran-GTP dependent manner. 17 In the cytoplasm, pre-miRNAs are further processed by the RNase III Dicer to yield ~21-25 nucleotide-long mature miRNAs. 17 They bind to the 3' untranslated region (UTR) of the target messenger RNAs (mRNAs) via partial sequence complementarity and subsequently lead to translational inhibition and mRNA decay of the target genes. 13 MiRNAs play key roles in development, as evident from early mouse model studies that knockout of miRNA processing proteins causes embryonic lethality. 18 Over the past two decades, miRNAs have also been associated with many dental pulp disorders and diseases, with many studies shedding light on their potential diagnostic and prognostic values. 19  Although a large number of miRNAs have been identified in apical periodontitis, only few were further studied and characterized. For example, Yue, et al. 22 (2016) found that miR-155 played a key role in inflammation and the progression of apical periodontitis by inhibiting SEMA3A. In another study, Yue, et al. 23 (2017) revealed that miR-335-5p acted as a positive mediator in human periodontal ligament fibroblasts (HPDLFs) inflammation and identified two targets, uPAR and RANKL. They found that uPAR was repressed by miR-335-5p at the basal level, but when HPDLFs were subjected to LPS stimulation, it could be relieved from miR-335-5p-mediated repression, a process called derepression; whereas miR-335-5p promoted RANKL in HPDLFs regardless of whether or not it was under inflammatory conditions. 23 Lina,et al. 24 (2019)  Not only did these findings confirm the diverse and complex miRNA regulatory network in apical periodontitis, but they also indicated that a few potential areas of study may be conducted in the future to further advance our understanding of miRNAs.

MicroRNAs in apical periodontitis
First, differential expression patterns of miRNAs may be cell type-specific. As mentioned before,  Figure   1). This will greatly enhance the informed decisionmaking process, and patient-centered treatment options can also be better tailored and rendered.
In addition, if miRNA profiling can be further stratified based on symptomatology (symptomatic vs. asymptomatic) or severity of the symptoms, this

MicroRNAs as therapeutic targets for apical periodontitis
Similar to the idea of biomarkers, miRNAs can also be designed as therapeutic targets. 31 As hinted at above, inhibitors of pro-inflammatory miRNAs or mimics of anti-inflammatory miRNAs can be used in conjunction with traditional endodontic therapies.
This type of treatment aims to prevent or inhibit the inflammatory process in apical periodontitis ( Figure   1). It was recently demonstrated that miR-10a-5p may suppress inflammation in apical periodontitis. 30 If this can be further shown with in vivo studies, then miR-10a-5p has the potential to be developed as a therapeutic agent and be used as an adjunct therapy along with the conventional endodontic treatment.  As many novel miRNAs are being unveiled, they have the potential to be translated into chairside diagnostic biomarkers and therapeutic agents. For example, before any endodontic treatment, pulpal/periapical fluid and blood can be easily sampled using instruments such as syringes or microcannulas, from which miRNA levels can be assessed. Based on the differential expression patterns of specific miRNAs that are associated with characteristics and/or prognostic factors of apical periodontitis, informed decision-making can be achieved, and patient-centered treatment options can be tailored and rendered. On the other hand, if specific miRNAs are known to play a role in pro-and anti-inflammatory process, their inhibitors/mimics can be applied to inflamed pulp tissues in conjunction with conventional vital pulp therapies such as direct pulp capping or delivered to the root canals and periapical regions using microcannulas during root canal therapies to promote better healing outcomes. Author disclosure statement