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Study on the Mechanism of miR-146a in Gingival Mesenchymal Stem Cells

This study aimed to investigate the molecular mechanisms of microRNA-146a (miR-146a) on gingival mesenchymal stem cells (MSCs). Gingival MSCs were isolated from the gingiva tissues of patients with periodontal disease to reveal the function of miR-146a in regulating osteoblast differentiation. miR-146a inhibits osteoblast differentiation by inhibiting phosphorylated cyclic-AMP response binding (CREB) protein translocation into the nucleus and ultimately attenuating runt-related transcription factor 2 (Runx2) expression. Furthermore, silencing miR-146a promotes the proliferation of gingival MSCs. Of note, targeted inhibition of miR-146a also inhibited LPS-induced inflammatory response and promoted the proliferation of gingival MSCs via CREB/Runx2 axis. MiR-146a is a key negative regulator of gingival MSCs proliferation and osteogenic differentiation, and targeting to reduce the miR-146a expression is essential for bone formation signaling. Therefore, we propose that miR-146a is a useful therapeutic target for the development of bone anabolic strategies.


Introduction
Periodontal disease is the most common oral disease, including categories of gum disease and periodontitis with deep periodontal tissue lesions, considered to be the main cause of tooth loss in adults, as well as the most signifcant oral disease that endangers human dental and even systemic health [1]. Periodontitis is mainly a chronic infammatory disease of periodontal supporting tissues caused by bacteria and is highly prevalent worldwide [2,3]. Currently, the promotion of dental bone and alveolar bone regeneration is considered to be the most promising treatment strategy for periodontitis [4,5]. Remodeling periodontal connective tissue and promoting bone and alveolar bone regeneration have been considered the most promising treatment strategy for periodontitis. Unfortunately, due to the complexity and specifcity of the pathogenesis of periodontitis and the periodontal microenvironment, conventional treatments are unable to completely repair the periodontal tissue damage caused by periodontitis [6]. Notably, with the maturation of stem cell and biological tissue engineering technologies (e.g., guided tissue regeneration, GTR), stem cells, especially periodontal stem cells, were considered to be the most reliable method to promote dental bone regeneration for the treatment of periodontitis and an important link to identify ideal seed cells [7].
As a primitive cell population with self-renewal and multidirectional diferentiation potential, MSCs, the cells of origin for the formation of various tissues and organs, have emerged as efective alternative cell sources for tissue engineering. Stem cells from the bone marrow and dental marrow have been shown to have a role in promoting periodontal osteogenesis [8,9]. Dental-derived MSCs were extracted from the dentition and its accessory tissues.
However, these MSCs are usually limited in their general application by the small number of dental-derived MSCs, the invasive process of obtaining them, and the difculty of their proliferation and diferentiation in vitro. Terefore, gingiva MSCs, which have properties easily obtained via minimally invasive cell isolation techniques, phenotypic stability, and multidirectional diferentiation potential, have attracted wide attention in the application of periodontal disease prevention and treatment. Numerous studies have shown that gingiva MSCs are crucial for tissue regeneration and immunomodulation, and their ability to promote periodontal bone regeneration has been demonstrated in animal models [10,11].
MicroRNA (miRNA), a class of small noncoding RNA sequences consisting of approximately 22 nucleotides, can regulate eukaryotic gene expression and infuence the synthesis of proteins encoded by target genes, thus participating in almost all cellular life activities, including cell proliferation, diferentiation, metabolism, immune regulation, infammatory response, and cell death. Numerous studies implied that miRNAs have played key roles in various aspects of periodontal tissue injury and repair, infammatory response, and homeostasis of the periodontal microenvironment, including promoting periodontal stem cell diferentiation and regulating osteoblast and osteoclast functions [12,13].
Previously, miR-146a, miR-143-3p, and miR-1226 have been reported to be positively associated with the progression of chronic periodontitis as well as other periodontal diseases [14,15]. Previous studies showed that miR-146a could promote angiogenesis in human umbilical vein endothelial cells (PMID: 28337286), regulate the function of T17 cell diferentiation to modulate cervical cancer cell growth and apoptosis (PMID: 30864722) and mitochondrial function, and cardiomyocyte apoptosis (PMID: 33717647). In addition, miR-146a negatively regulates osteogenesis and bone regeneration in adipose-derived mesenchymal stem cells (PMID: 28205638). Of particular note, miR-146a has been shown to be a potential therapeutic target for chronic periodontitis by inhibiting the chronic infammatory response and immunomodulatory efects of periodontal tissues [17]. However, the role and molecular basis of miR-146a-regulated osteogenic diferentiation of gingiva MSCs remain unclear, which greatly limits its clinical application and transformation as a therapeutic target. Here, we mechanically evaluated the efects of miR-146a on cell proliferation and osteogenesis of gingiva MSCs. Our fndings suggest that miR-146a plays a pivotal role in gingival mesenchymal stem cell proliferation and osteogenic differentiation. Tus, targeting to reduce the miR-146a expression is essential for bone formation signaling, which may be a new early warning and therapeutic target for chronic periodontitis. . MiR-146a inhibitor and miR-146a mimics were obtained from Shanghai GenePharma Co., Ltd. All other chemicals were of analytical grade.

Gingiva
MSCs Isolation and Culture. Te gingiva was obtained from patients with periodontal diseases. Te volunteers were admitted to the periodontal department of the Zijingang branch of the afliated stomatology hospital, Zhejiang University School of Medicine. All procedures are approved by the clinical research ethics committee of Zhejiang University School of medicine and informed consent was provided 2021-88 (R). Te gingiva MSCs were isolated and cultured as previously described [18]. Gingiva MSCs were cultured in α-MEM with 15% fetal bovine serum (FBS) supplemented with 1% (v/v) penicillin and streptomycin in a constant environment of 37°C/5% CO 2 . All cells were harvested after specifc treatments for further investigation.

Infection of miR-146a
Overexpression. We insert miR-146a and sponge sequence into a pLVX-H1-GFP-puro cloning vector (GenePharma) to generate lentivirus. Te PCR primers are provided in Supplementary Table 2. Transfection of gingiva MSCs with the plasmid of miR-146a overexpression and sponge to receive lentivirus for the subsequent experiment [19]. Te transfection efect was observed using fuorescence microscopy [20].

Gingiva MSCs Viability
Test. Gingiva MSCs were seeded in 96-well plates with 4 × 10 3 cells/well. Gingiva MSCs were transfected with miR-146a overexpressing lentivirus and cultured continuously for 7 days, and cells transfected with an overexpressing empty plasmid were used as control. Te proliferation activity of gingiva MSCs was detected daily using a fat plate clone formation test and CCK-8 assay according to the vendor's instructions.

Osteoblast and Osteoclast Diferentiation.
Gingiva MSCs plated in 6-well plates were maintained in an α-MEM medium containing 10% FBS, 10 mM dexamethasone (Sigma), and 10 mM β-glycerophosphate (Alfa Aesar), 50 μM Vitamin C (Sigma) and 1% antibiotic/antimycotic. Gingiva MSCs were continuously cultured for 4 weeks before applying von Kossa staining to detect mineralized nodules. Medium changes are done every 4 days during the culture. Meanwhile, gingiva MSCs were cultured in α-MEM containing 10% FBS and 50 ng/ml RANKL (R&D), and 25 ng/ml M-CSF (Peprotech) to assay the osteoclastogenesis. After 10 days of culture, the gingiva MSCs were subjected to PBS wash and then fxed in 4% paraformaldehyde solution for 10 minutes before TRAP (anti-tartrate acid phosphatase) staining, and fnally observed under an inverted microscope. Osteoblasts and osteoclast were cocultured with the miR-146a overexpression plasmid in 6-well plates for 7 days, respectively. Subsequently, the expression levels of relevant genes and proteins in osteoblasts and osteoclasts were measured.

Quantitative Real-Time PCR.
To further verify the results, real-time PCR was employed to quantify relative mRNA expression levels. First, the mRNA was extracted from cells by Trizol (TAKATA, Osaka, Japan) and reversetranscriptional reactions were performed using a TAKATA Reverse Transcriptase kit (TAKATA, Osaka, Japan). Removal of DNase and cDNA reverse synthesis and real-time PCR were then performed as protocol. Te qPCR reaction mixture was prepared using SYBR Green Master Mix (Tiangen, Beijing, China) and then run on a CFX Con-nectTM Real-Time PCR System (Bio-Rad, California, USA) as previously described. U6 snRNA was used as an internal reference for miR-146a. GAPDH was used as an internal control for other gene assays in this study. Te qPCR primers are provided in Supplementary Table 2. 2.9. Western Blot. Western blot was commonly used to detect the protein expression level of cells. In short, proteins to be tested were extracted from cells and were separated within SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred into the polyvinylidene fuoride (PVDF) membranes. After blocking and incubation of corresponding antibodies (Supplementary Table 1) for 12 h, specifc binding with second antibodies was performed, and the immunoblotting of target protein bands were observed using a ECL chemiluminescence and gel documentation system (G: BOX F3, Gene). Comparisons between two groups and among three or more groups were achieved using the two-tailed unpaired t-test or analysis of variance (ANOVA), respectively. P < 0.05 was considered statistically signifcant.

Characterization of Gingiva MSCs from Gingiva with
Periodontitis. Macroscopically, gingiva MSCs from gingiva with periodontitis appeared with a spindle fbroblast-like morphology under light microscopy, similar to other types of MSCs (Figure 1(a)). Subsequently, the abundance of surface markers in gingiva MSCs was determined by fow cytometry and immunofuorescence. As shown in Figure 1(b), CD29, CD105, and CD90 showed a positive expression and CD34 showed negative expression in gingiva MSCs. Meanwhile, we used Oil Red O staining to test the diferentiation potential of gingiva MSCs. Moreover, lipid accumulation was observed in the cytoplasm of those differentiated gingiva MSCs after 2 weeks of culture in a lipogenic diferentiation medium, indicating existed diferentiated gingiva MSCs (Figure 1(c)). In contrast, no positive signals were observed in the cells cultured with the standard medium. Tese results confrm that cultured gingiva MSCs from gingiva with periodontitis possess stem cell properties.

Silencing miR-146a Attenuates LPS-Mediated Infammatory Response in Gingiva
MSCs. Chronic infammation of the periodontal tissue from the deep gingival layer to the periodontium and alveolar bone is a key factor in triggering periodontitis [21]. Infammatory factors released by periodontal tissue cells, such as TNF-α, IL-6, and IL-1β, maintain the periodontal infammatory environment for a long time and can lead to irreversible periodontal tissue Evidence-Based Complementary and Alternative Medicine damage [22]. Terefore, we further assessed the regulatory role of miR-146a on the infammatory response of gingiva MSCs. First, we examined the content and expression levels of infammatory factors in gingiva MSCs overexpressing miR-146a. Te results show that elevated miR-146a significantly promotes the secretion of infammatory factors (Figures 3(a)-3(c)), and also increases the mRNA levels of TNF-α, IL-6, and IL-1β in gingiva MSCs (Figure 3(d)). Studies reveal that local infammatory responses can exacerbate periodontal tissue damage and accelerate the progression of periodontitis [21]. Notably, silencing miR-146a in the LPS-mediated infammatory response model of gingiva MSCs signifcantly decreased the mRNA and protein abundance of infammatory factors and reduced their release (Figures 3(e)-3(h)). Tese data suggest that miR-146a can drive the infammatory response in gingiva MSCs.

Efects of miR-146a on the Osteogenic Diferentiation of
Gingiva MSCs. Te promotion of osteogenic diferentiation of MSCs is considered the most promising therapeutic strategy for chronic damaging diseases of bone tissue in clinical settings, such as osteoporosis and osteoarthritis. As shown in Figure 4(a), gingiva MSCs were cultured for 4 weeks, and mineralized nodules were visualized using von Kossa staining after transfection with a control plasmid, but not visualized with miR-146a overexpression plasmid. Cells were cultured in the medium for osteoclasts for 10 days and osteoclast was detected by TRAP staining. Similarly, TRAPpositive staining was observed in cells after transfected with a control plasmid but was almost not visualized with the miR-146a overexpression plasmid. Meanwhile, the result of coculture showed that miR-146a signifcantly decreased the expression of several core binding factors of osteogenic diferentiation in hepatocytes, including bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), collagen type I collagen α1 (COL1α1), osteopontin (OPN), and osteocalcin (OCN) (Figure 4(b)). Consistently, miR-146a severely reduced the expression levels of the osteogenic key proteins (BMP2, COL1α1, OPN, and OCN) in gingiva MSCs. Briefy, these data indicated that miR-146a can inhibit osteogenic key enzyme expression to suppress osteogenesis in gingiva MSCs (Figures 4(c) and 4(d)).

miR-146a Regulates Osteogenesis in Gingiva MSCs via the CREB/Runx2
Axis. cAMP-response element-binding protein (CREB), an important transcriptional regulator, Evidence-Based Complementary and Alternative Medicine efectively promotes the osteogenic diferentiation of MSCs [23]. Analyzing the interaction of miR-146a with target genes by target prediction database miRDB and miRcode, we found that CREB strongly interacts with miR-146a ( Figure 5(a)). By overexpressing and/or silencing miR-146a, we confrmed that the expression of CREB is modifed by miR-146a which in turn regulates osteogenesis in gingiva MSCs. Runt-related transcription factor 2 (Runx2) was a crucial regulator in promoting bone formation, which has important regulatory roles in osteoblast diferentiation, chondrocyte maturation, and bone metabolism. First, we found that the overexpression of miR146 signifcantly attenuated CRBE/RUNX2-mediated osteogenic signaling in gingiva MSCs (Figures 5(b) and 5(c)). However, the overexpression of CREB was able to promote RUNX2 transcriptional regulation to restore miR-146a-mediated reduction in osteogenic key factor expression ( Figures 5(d)-5(f )). Tese results reveal that miR-146a inhibits the osteogenic potential of gingiva MSCs in vitro by regulating the CREB/Runx2 axis.

Discussion
Te osteogenic capacity of seed cells is an important indicator of periodontal tissue regeneration. Promoting the osteogenic potential of seed cells has been considered an Evidence-Based Complementary and Alternative Medicine important tool for periodontal osteogenesis and remodeling the physiological barrier of periodontal tissues [24]. With the advancement of tissue engineering technology, MSCs are widely used in clinical applications. Even though MSCs have multiple sources, gingiva MSCs have more distinct advantages due to their easy availability during routine dental procedures and the absence of signifcant diferences in the characteristics of gingiva MSCs from healthy gingival tissues and those from hyperplastic or infamed gingival tissues [25]. It has been shown that MSCs characteristics isolated from gingival tissues can exhibit a stable phenotype and have the potential for multidirectional diferentiation during long-term culture [26]. Recent epidemiological surveys show that more than half of adults worldwide sufer from gingivitis and periodontitis, with a trend toward younger adults [27]. Clinically, the main goal of periodontitis treatment is to inhibit plaque and eliminate infammation to prevent deterioration and recurrence of the disease [28]. Given the potential for directed diferentiation of MSCs, using gingiva MSCs was expected to be an efective solution for the radical treatment of periodontitis [29]. Our previous study confrmed the osteogenic ability of gingiva MSCs, but the amount of new bone in rat bone defects was limited [26], so we tried to explore efective avenues to improve the osteogenic ability of gingiva MSCs and elucidate the molecular mechanism in this study. MicroRNA, a noncoding single-stranded RNA regulated by posttranscriptional gene expression, plays an extremely important role in the infammatory response, immune response, and almost all aspects of periodontal tissue, including periodontal stem cell diferentiation, osteoblast function, and osteoclast function [30,31]. miR-146a is one of the frst miRNAs identifed to be involved in the infammatory response and is abnormally and signifcantly highly expressed in periodontal infammatory tissues, promoting the development of periodontitis [31]. miR-146a has been reported to promote diferentiation in periodontal ligament cells through the downregulation of NF-kappa B signaling (PMID: 20110513). In this study, we demonstrated that highly expressed miR-146a in periodontitis patient-derived gingiva MSCs can impair their osteogenic potential, block the cycle distribution of gingiva MSCs, and induce apoptosis. Similarly, the miR-146a overexpression drove infammatory factor release in gingiva MSCs mediating the onset of infammatory responses, and targeted inhibition of miR-146a signifcantly attenuated LPS-mediated infammatory responses in gingiva MSCs. Interestingly, the targeted intervention of miR-146a activates Runx2 transcriptional regulation of the expression of key proteins (BMP2, OPN, OC, and BSP) of osteogenic signaling. Runx2 directly stimulates the transcription of osteocalcin, type I collagen, bone-bridging protein, and collagenase 3 genes to promote osteogenic diferentiation during the diferentiation of bone marrow mesenchymal cells to osteoblasts [32]. Bone morphogenetic protein (BMP), a target gene of Runx2 transcriptional regulation, is the most efective inducer of osteoblast diferentiation and bone formation to regulate osteogenic diferentiation [33]. BMP promotes osteogenic diferentiation mainly by binding to heteromeric receptor complexes [34]. Evidence suggests that CREB is recruited to CRE in the Sma6 promoter and enhances the Smad6 expression and thus CREB regulates RUNX2 transcriptional activation was an important pathway to promote osteogenic diferentiation [35]. As a "bridge" between the bone system and immune system, the CREB/Runx2 axis is a key factor to determine alveolar bone resorption or regeneration in the process of periodontitis development, and to a large extent afects the prognosis and outcome of periodontitis. To further elucidate the molecular mechanism of   whether the same mechanism occurs in animal models of periodontitis or even in the periodontitis population remains to be answered. In follow-up studies, we will further verify the results of the study in vivo through animal model tests. Also, we know that most infammatory mediators are produced by macrophages in the infammatory environment. MSCs have been reported to skew LPS-stimulated macrophage polarization towards the M2-like phenotype   and reduce infammatory reactions by secreting TGF-β cytokines (PMID:31771622). However, whether the efect of miR-146a on infammation is caused by macrophage polarization needs to be further investigated.

Conclusion
In summary, our fndings suggest that miR-146a plays an important role in the osteogenic diferentiation of gingiva MSCs through the CREB/Runx2 axis. miR-146a is a key negative regulator of gingival mesenchymal stem cell proliferation and osteogenic diferentiation and targeting to reduce miR-146a expression is essential for bone formation signaling. Terefore, we propose that miR-146a holds promise as an attractive therapeutic target for periodontitis treatment.

Data Availability
Te data and materials used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.