The Expression and Regulatory Roles of Long Non-Coding RNAs in Periodontal Ligament Cells: A Systematic Review

Periodontal ligament (PDL) cells play a pivotal role in periodontal and bone homeostasis and have promising potential for regenerative medicine and tissue engineering. There is compelling evidence that long non-coding RNAs (lncRNAs) are differentially expressed in PDL cells compared to other cell types and that these lncRNAs are involved in a variety of biological processes. This study systematically reviews the current evidence regarding the expression and regulatory functions of lncRNAs in PDL cells during various biological processes. A systematic search was conducted on PubMed, the Web of Science, Embase, and Google Scholar to include articles published up to 1 July 2021. Original research articles that investigated the expression or regulation of lncRNAs in PDL cells were selected and evaluated for a systematic review. Fifty studies were ultimately included, based on our eligibility criteria. Thirteen of these studies broadly explored the expression profiles of lncRNAs in PDL cells using microarray or RNA sequencing. Nineteen studies investigated the mechanisms by which lncRNAs regulate osteogenic differentiation in PDL cells. The remaining 18 studies investigated the mechanism by which lncRNAs regulate the responses of PDL cells to various stimuli, namely, lipopolysaccharide-induced inflammation, tumor necrosis factor alpha-induced inflammation, mechanical stress, oxidative stress, or hypoxia. We systematically reviewed studies on the expression and regulatory roles of lncRNAs in diverse biological processes in PDL cells, including osteogenic differentiation and cellular responses to inflammation, mechanical stress, and other stimuli. These results provide new insights that may guide the development of lncRNA-based therapeutics for periodontal and bone regeneration.


Introduction
Periodontitis is a plaque-induced inflammatory oral disease that causes the progressive breakdown of periodontal tissue, and it is one of the leading causes of tooth loss [1,2]. Although conventional therapies can control active periodontal inflammation, they are unable to fully regenerate damaged periodontal tissue. Therefore, recent efforts to treat periodontal diseases have focused on regenerative therapies that can restore the physiological function of teeth by re-building supporting periodontium, including periodontal ligament (PDL), alveolar bone, gingiva, and cementum [3,4].
PDL is a thin layer of fibrous connective tissue, located between the alveolar bone and cementum, that plays a crucial role in the development, functioning, and regeneration of the tooth-supporting apparatus. An early study of PDL found that it had a regenerative capacity and possibly contained a population of multipotent progenitor cells [5]. It has since been established that PDL cells are a heterogeneous cell population consisting of fibroblastic and osteoblastic mesenchymal lineages that include cells at different stages of

Selection Criteria
The inclusion criteria were as follows: (1) studies based on cell, human, or animal models; (2) studies related to the expression or regulation of lncRNAs in PDL cells; and (3) studies published in English. The exclusion criterion was as follows: reviews, conference abstracts, or editorials.

Selection of Studies
Titles and abstracts of manuscripts were independently screened in electronic sheets by two reviewers (Y.L. and Z.T.). Titles and abstracts were examined, and duplicate studies were eliminated. If an article's abstract did not contain sufficient information for an inclusion/exclusion decision to be made, its full text was obtained and carefully inspected. Any inter-examiner disagreement was resolved by discussion. The level of agreement between the two examiners was assessed by determining Cohen's kappa scores.

Quality Assessment
The quality of selected papers was evaluated using a well-known system (Table S1) described by Wells and Littell [34]. The following eight questions comprised the quality scoring system. (1) Was the study hypothesis/aim/objective clearly described? (2) Were the experimental designs in the study well described? (3) Were the methods and materials in the study well described? (4) Were the time-points of data collection in the study clearly defined? (5) Were the main outcomes of measurements in the study clearly defined? (6) Were the experimental groups comprehensively compared with the control group in the study? (7) Were the results in the study well described? (8) Were the limitations of the study discussed? In answering each question, 1 point was allocated for "yes" and 0 points were allocated for "no." The sum of scores for each study was calculated independently, and the total possible score was 8. A score of 7 to 8 indicates a study with excellent quality, a score of 5 to 6 indicates a good quality study, a score of 3 to 4 indicates a low-quality study, and a score of 0 to 2 indicates a bad quality study. A detailed evaluation of the scores of selected studies is presented in Table S1.

Literature Search and Screening of Studies
A flow diagram of study selection is shown in Figure 1. Two hundred and eighty records were obtained by screening titles and abstracts and removing duplicates. After reviewing these titles and abstracts, 73 articles were retrieved for full-text evaluation, and 23 were subsequently excluded for the reasons described in the diagram. The remaining 50 studies were included for further analysis. The kappa score for study selection was 0.939, indicating that there was an excellent level of agreement between the reviewers. All studies were published between 2014 and 2021, and their characteristics are summarized in Figure 2.

Discussion
This study systematically reviewed studies exploring the expression of lncRNAs and their role in the regulation of a variety of biological activities in PDL cells, such as osteogenesis and cell response to inflammation and mechanical stress. LncRNAs regulate gene expression at transcriptional and post-transcriptional levels. At the transcriptional level, lncRNAs may directly bind to DNA or act on transcriptional complexes, resulting in cis or trans gene activation or silencing [78,79]. LncRNAs can recognize and bind to complementary RNA sequences, which enables highly specific interactions that can regulate various post-transcriptional processes, such as mRNA splicing, transport, translation, and stabilization, thereby affecting various biological processes [78,80]. LncRNAs can also specifically recruit and integrate with RNA binding proteins (RBPs) to regulate their biological functions, thereby affecting the expression of downstream genes [81]. In addition to regulating mRNAs via independent mechanisms, lncRNAs can act as competing endogenous RNAs (ceRNAs) by competitively binding to miRNAs via miRNA response elements. This binding attenuates the ability of miRNAs to downregulate mRNA expression and thus indirectly regulates mRNA expression [82]. LncRNA-mediated ceRNA interactions have been identified in various cancers and inflammatory diseases, including periodontitis [83][84][85].

Studies on lncRNA Expression Profiling in PDL Cells
In the 13 studies that explored lncRNA expression profiles in PDL cells, 6 used microarray analysis and 7 used RNA-seq methods [27][28][29][30][35][36][37][38][39][40][41][42][43]. Recent developments in RNA-seq techniques offer enormous potential for transcriptome characterization as they are reliable tools for elucidating genetic and metabolic pathways involved in biological processes. RNA-seq provides more comprehensive information about the characteristics of transcripts as this information is not limited to the known genes represented on a microarray and novel transcription variants can be detected via alternative splicing [86].
Three of these thieteen studies compared lncRNA expression profiles in PDLSCs with the lncRNA expression profiles of other cell types, such as BMSCs, GMSCs, and DFCs [27,42,43]. Moreover, 5 of these 13 studies examined the lncRNA expression profiles of PDLSCs under osteogenic induction [28][29][30]35,36]. The results varied, showing differently up-and downregulated lncRNAs during the osteogenic differentiation process. These variations may be attributable to differences between samples and periods of induction. For example, Qu et al. [35] and Zhang et al. [29] examined three osteogenic-induced and three non-induced samples, whereas some authors did not mention the number of samples tested, and the period of osteogenic induction varied widely (3, 5, 7, or 14 days) between studies. It has been suggested that aging can affect the characteristics of the regenerative potentials of dental-derived stem cells [87,88]. Moreover, differences in library preparation, sequencing techniques, and methods of analysis may also have led to the variations in the results. Five of the thirteen studies explored the lncRNA profile of PDL cells subjected to mechanical stress [37][38][39][40][41]. Three of these studies applied tensile force on cells (10% or 12% equibiaxial strain) [37,39,40] and one applied compressive force on cells (2 g/cm 2 ) for 12 h [38]. After microarray or RNA-seq, most studies performed only PCR to validate the expression of several genes. Further in-depth studies are warranted to explore the regulation of the identified lncRNAs.
TUG1 was initially identified as an important gene in retinal development and the formation of photoreceptors [89]; later, it was reported to be abnormally expressed during tumorigenesis [90]. It was observed that TUG1 can bind to lin-28 homolog A, an RBP, thereby promoting the expression of osteogenesis-related markers and the osteogenic differentiation of PDLSCs [32]. Wu et al. reported a post-transcriptional regulatory mechanism by which TUG1 enhanced the osteogenic differentiation of PDLSCs: TUG1 sponges microRNA-222-3p, which promotes osteogenic differentiation by upregulating Smad 2/7, which are the main signal transducers for receptors of transforming growth factor beta. The knockdown of TUG1 or overexpression of microRNA-222-3p inhibited this upregulation [55]. MEG3, initially known as a tumor suppressor, is another lncRNA that has received much attention due to its association with the osteogenic differentiation of MSCs, DFCs, and PDL cells [33,49,91,92]. In PDL cells, MEG3 attenuates bone morphogenetic protein 2 (BMP2) expression by competing with BMP2 for binding to the RBP heterogeneous nuclear ribonucleoprotein I [49]. Furthermore, four studies have investigated the regulatory role of lncRNAs in the osteogenesis of P-PDL cells isolated from the extracted teeth of patients with periodontitis [33,45,50,58]. P-PDL cells were first isolated in 2010 and have since attracted much attention [93]. However, P-PDL cells have been shown to have less osteogenic differentiation potential than H-PDL cells [94][95][96][97]. Wang et al. used microarray analysis to identify a novel lncRNA, lncRNA-POIR, which is differentially expressed in P-PDLSCs. They found that LncRNA-POIR regulates Forkhead box O (FOXO)1 by sponging miR-182 and, thus, inhibits the canonical Wnt pathway and promotes osteogenesis [45].
LPS is an endotoxin and a major component of the cell membranes of Gram-negative bacteria, such as Porphyromonas gingivalis and Escherichia coli, where it performs various biological activities. It is mediated by the toll-like receptors (TLR) 2, and TLR4 and triggers cytokine-mediated immune-inflammatory responses in the host, which results in the release of a wide range of pro-inflammatory cytokines. Several lncRNAs, including TUG1, MEG3, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), FGD5-antisense RNA 1 (FGD5-AS1), and LINC01126, have been reported to modulate the inflammatory response of PDL cells to LPS challenge. Huang et al. and Han et al. reported that the expression of TUG1 is decreased in PDL cells upon LPS challenge, but they ascribed this to different regulatory mechanisms [67,71]. Han et al. reported that TUG1 competes with miR-132 to promote the proliferation and inhibit the apoptosis of PDL cells under inflammatory stimuli [67]. More recently, Huang et al. suggested that TUG1 is a sponge of miR-498, which allows it to regulate the expression of RAR-related orphan receptor A and attenuate LPS-induced activation of the Wnt/beta-catenin pathway [71].
There has been extensive research on the regulatory mechanisms of orthodontic tooth movement. PDL cells subjected to mechanical stress are widely used to mimic in vivo conditions. Three lncRNAs, DANCR, MIR31 host gene (MIR31HG), and FER1L4, have been investigated for their role in the regulation of compressive force-induced biological activities in PDL cells [73][74][75]. It was suggested that the knockdown of DANCR inhibits the osteoclast formation and root resorption that is induced by compressive force via miR-34a-5p/jagged1 [73]. In addition, lncRNAs also regulate force-induced autophagy in PDL cells. For example, FER1L4 mediates compression-induced autophagy via the AKT/FOXO3 signaling pathway [75]. Notably, these studies have focused only on the effects of compressive stress on the regulation of lncRNAs in PDL cells; there have been no investigations on the effects of other types of stress loadings, such as tensile or shear forces, on the regulation of lncRNAs in PDL cells.

Future Perspectives
Non-coding RNAs possess critical biological functions that were initially discovered in cancer research and then in stem cell studies, and an increasing number of lncRNAs have been discovered in the field of regenerative medicine. With the rapid development of high-throughput sequencing, it is critical to screen diverse lncRNAs and further investigate their roles in various biological functions. With more in-depth research, lncRNAs and their target genes may be identified as possible therapeutic targets in clinically relevant diseases. This review summarizes current research on lncRNAs in PDL cells, with a focus on the expression profile of lncRNAs, their regulation of osteogenic differentiation and the effect upon stimulations. However, most of these recent developments are still in the in vitro stage, and clinical application remains a challenge.

Conclusions
PDL cells have significant potential for use in the clinical application of periodontal and bone regeneration. This study systematically reviewed studies exploring the expression and regulatory roles of lncRNAs in the diverse biological processes of PDL cells, such as osteogenic differentiation and cellular responses to inflammation, mechanical stress, and other stimuli. However, most of these studies were focused on in vitro analyses; more in vivo investigations are required in this promising translational field.
Author Contributions: Y.L. conceptualized the topic, collected the data, performed the statistical analysis, and wrote the manuscript. Z.T. collected the data, performed the statistical analysis, and wrote the manuscript. L.J. conceptualized the topic and supervised and facilitated the conduct of the study. Y.Y. participated in all stages of the review and supervised the conduct of the study. All authors critically revised the manuscript for important intellectual content and approved the final version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.