MicroRNA-27a-5p Downregulates Expression of Proinflammatory Cytokines in Lipopolysaccharide-Stimulated Human Dental Pulp Cells via the NF-κB Signaling Pathway

MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). LPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated the expression of proinflammatory cytokines, NF-κB activity, and the expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3’-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.


Introduction
Pulpal inflammation is predominantly induced by dental caries-associated bacteria and their related products.Local immune responses in this tissue manage these microbiological stimuli [1].Dental pulp cells close to infected dentinal tubules are the first defense against bacterial stimuli.Prolonged bacterial invasion into pulp tissue induces the infiltration of specific immunocompetent cells, such as macrophages, dendritic cells, and lymphocytes [2].Odontoblasts and dental pulp fibroblasts express Toll-like receptor (TLR) 4 and TLR2 [3,4], which function as pattern-recognition receptors that bind to lipopolysaccharide (LPS), a typical pathogen-associated molecular pattern [5].Stimulation of TLR2/4 by LPS activates several signaling pathways, including nuclear factor-κB (NF-κB), in dental pulp cells and subsequently induces the synthesis of proinflammatory mediators, including interleukin (IL)-1β, tumor necrosis factor α (TNFα), IL-6, IL-8, and monocyte chemotactic protein 1 (MCP1) [5][6][7][8].These infection-induced inflammatory molecules are involved in eradicating the invading microbes, but, concurrently, they have the potential to induce collateral tissue damage [9].Excessive or longstanding inflammatory reactions can induce severe pulp tissue damage that may result in total pulp necrosis.Conversely, a relatively mild inflammatory response encourages tissue regeneration by activating dental pulp stem cell (DPSC) migration, proliferation, and differentiation [10][11][12].Low-grade inflammation might play a pivotal role in initiating early reparative or regenerative processes [13].Therefore, it is important to regulate the inflammatory and immune responses of dental pulp at an appropriate level.
MicroRNAs (miRNAs) are a class of non-coding RNAs of approximately 23 nucleotides and play crucial roles in the negative regulation of post-transcriptional gene expression.They bind to the 3 ′ -untranslated region (UTR) to induce degradation of target mRNAs and impede translation [14,15].MiRNAs have an important effect on various biological processes, such as cell proliferation, survival, apoptosis, and inflammation [16,17] Increasing evidence has revealed that the expression levels of certain miRNAs vary during inflammatory responses [18].In human leukemic cells, miR-146a is upregulated upon LPS binding to TLR 2/4 and subsequent NF-κB activation, and the promoted miR-146a downregulates proinflammatory cytokines by targeting activators of NF-κB signaling, such as tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1) [19].A reduction in miR-223 is presumably involved in the promotion of irreversible pulpitis in human dental pulp tissue because miR-223 negatively regulates the production of IL-1β and IL-18 via the miR-223/NLRP3/CASP1 axis in human dental pulp fibroblasts [20].Moreover, miR-27a has a critical role as an anti-inflammatory mediator in acute lung injury [21], the spinal cord [22], human articular chondrocytes [23], and human aortic endothelial cells [24] by targeting multiple genes.Our previous study evaluated LPS-induced miRNA expression changes in human dental pulp cells (hDPCs) compared to non-stimulated cells using a miRNA array and found that miR-27a-5p was among highly upregulated miRNAs with a 3.14-fold increase (Table S1).However, much remains to be clarified concerning the regulatory role of miR-27a-5p in dental pulp inflammation.Therefore, this study investigated how miR-27a-5p regulates proinflammatory cytokine expression in LPS-stimulated hDPCs, focusing on the LPS/TLR4/NF-κB pathway as the intracellular signaling responsible for miR-27a-5p-induced regulation.

Discussion
Elevated miR-27a expression after exposure to LPS has been reported in rat pancreatic acinar cells [28], dairy cow mammary epithelial cells [29], and CD-1 mice [30].However, few studies have investigated the role of miR-27a in pulpal inflammation.In this

Discussion
Elevated miR-27a expression after exposure to LPS has been reported in rat pancreatic acinar cells [28], dairy cow mammary epithelial cells [29], and CD-1 mice [30].However, few studies have investigated the role of miR-27a in pulpal inflammation.In this study, we revealed significant upregulation of miR-27a-5p in hDPCs and rat incisor pulp tissues stimulated by LPS.LPS stimulation also upregulated the expression of proinflammatory cytokines, including IL-6, IL-8, and MCP1, in hDPCs, which occurred concurrently with the increase in miR-27a-5p expression.These findings indicate a shared upstream signaling pathway between miR-27a-5p and proinflammatory cytokines.TLRs, which are regarded as pattern recognition receptors, are crucial for immune responses, especially pathogen recognition [31].In particular, TLR2 and TLR4, expressed on human dental pulp cells [32][33][34][35][36], are essential for LPS detection, which is involved in triggering the transcription factor NF-κB, leading to the synthesis of various proinflammatory mediators [31,36].We verified that activation of NF-κB and its downstream mediators induced by LPS was negatively regulated in hDPCs by treatment with BAY 11-7085, an NF-κB inhibitor.Furthermore, LPS-stimulated miR-27a-5p expression was downregulated by BAY 11-7085, indicating that NF-κB signaling is upstream of both miR-27a-5p and proinflammatory mediators, as reported previously [37,38].The binding site of NF-κB p65 (RELA) is located in the miR-27a promoter region [37,38].
We investigated whether miR-27a-5p produced in LPS-stimulated hDPCs and pulp tissues exerts on the synthesis of proinflammatory mediators.The results revealed that overexpression of miR-27a-5p downregulated the expression of IL-6, IL-8, and MCP1 at both mRNA and protein levels in LPS-stimulated hDPCs and the expression of Il6 and Mcp1 mRNAs in LPS-applied rat pulp explants.These findings demonstrate miR-27a-5p as a negative regulator of proinflammatory cytokine synthesis, and it may act as a member of negative feedback loops.We have reported that miR-21 and miR-146b negatively regulate proinflammatory cytokine synthesis by targeting NF-κB in LPS-stimulated hDPCs [39,40], and miR-27a-5p may be a negative regulator in pulpal inflammation.Excessive synthesis of proinflammatory cytokines leads to the destruction of homeostasis, which induces severe damage to host tissues [41][42][43].Taken together, it is reasonable to propose that miR-27a-5p contributes to negative feedback networks in inflamed dental pulp to maintain the integrity of this tissue.
MiRNA regulates target genes through partial or complete complementary binding between the target gene 3 ′ -UTR and the miRNA seed base, suppressing target gene expression and subsequent protein synthesis [44].MiRNA-27a modulates multiple components of the TLR/NF-κB pathway, such as IRAK4, RELA [24,26], and FSTL1 [27].In this study, expression of these genes was attenuated through overexpression of miR-27a-5p in LPSstimulated hDPCs and rat incisor pulp tissues.We predicted TAB1 to be a candidate target of miR-27a-5p among NF-κB pathway components.TAB1 is required to initiate TGF-betaactivated kinase 1 (TAK1) activation, triggering several downstream inflammatory signaling pathways, including NF-κB, and inducing the synthesis of proinflammatory cytokines.Activation of TAK1 does not occur in TAB1 −/− mouse embryonic fibroblasts.The TAK1-TAB complex is reported to be closely related to multiple diseases [45,46].Knocking down TAB1 reduces cell hypertrophy of cardiomyocytes [47].Deficiency or mutations of TAB1 attenuate inflammation reactions induced by LPS, TNFα, or IL-1β stimulation [46,48,49].Expression of TAB1 was downregulated by miR-27a-5p in LPS-stimulated hDPCs and rat incisor pulp tissues.The luciferase reporter assays further revealed that the 3 ′ -UTR region of TAB1 contained the miR-27a-5p binding sequence.Knockdown of TAB1 led to decreased levels of phosphorylated NF-κB and IL-6, IL-8, and MCP1.Collectively, it was demonstrated that depletion of TAB1 significantly diminished NF-κB activation and the inflammatory response induced by LPS stimulation.These results provide a new insight into the regulatory mechanisms of miR-27a-5p in inflammatory reactions, particularly those involving TAB1, where miR-27a-5p downregulates the production of proinflammatory mediators by blocking the TAB1-NF-κB signaling pathway.
MiRNAs are being explored as therapeutic agents in cancer and various other fields [50].Exosomes, which are small vesicles released from cells, are under investigation as carriers for miRNA delivery due to their ability to modulate immune responses and target specific disease mechanisms [51].MiRNAs like exosomal miR-21 in hDPCs, colon cancer, and miR-15 and miR-16 in acute myeloid leukemia are promising therapeutic targets [50,51].Research also extends to lung cancer, retinal disorders, and toxicogenomics [50].Despite challenges such as rapid degradation and limited tissue penetration, clinical/preclinical trials are showing positive results [50].Our observations highlight the prospective application of miR-27a-5p as a treatment approach for pulp inflammation.
However, a limitation of this study lies in the absence of validity for miR-27a-5p detection in human pulpal inflammation in vivo.Further research is required to validate the effectiveness and safety of miR-27a-5p as a viable in vivo therapy, despite the positive results of ex vivo evaluations using rat dental pulp tissue and in vitro investigations using hDPCs.Moreover, additional studies are needed to determine the optimal methodologies to apply miR-27a-5p to inflamed human pulp tissue.

Establishment of the Pulpitis Model in Rats
Animal experiments were approved by the Animal Experimentation Committee of the Tokyo Medical and Dental University (A2019-297C).Six-week-old male Sprague-Dawley rats (Clea Japan, Tokyo, Japan) were fed a standard laboratory rat diet and allowed to drink water freely.The rats were anesthetized with 50 mg/kg ketamine hydrochloride (Parke-Davis, Detroit, MI, USA) and 20 mg/kg xylazine (Bayer, Leverkusen, Germany) injected intraperitoneally.Then, pulp tissue in maxillary and mandibular incisors was exposed using a round diamond bur, followed by application of 1 µL LPS in sterile saline (10 mg/mL) using sterile paper points.Sterile saline served as a negative control.The cavities were sealed with a light-curing resin (G-FIX; GC, Tokyo, Japan).The rats were euthanized by CO 2 inhalation at 0 (no pulp exposure), 3, 6, 12, and 24 h postoperatively.Following tooth extraction, extirpated pulp tissues were stored in RNAlater (Thermo Fisher Scientific) and subsequently analyzed for miR-27a-5p expression by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR).Rats were euthanized with CO 2 at 0 and 12 h for histological analysis.Upper and lower incisors were harvested, fixed in 4% paraformaldehyde in PBS at 4 • C for 24 h, and demineralized with EDTA for 14 days.The samples were then embedded in a tissue-embedding medium (Tissue-Tek O.C.T. Compound; Sakura Finetek, Tokyo, Japan) and sectioned into 10 µm slices using a cryostat (CM3050; Leica Microsystems, Wetzlar, Germany).These samples were stained with H&E.

Ex Vivo miR-27a Mimic Transfection
Male Sprague-Dawley rats (6 weeks old; Clea Japan) were euthanized by CO 2 inhalation, and their maxillary and mandibular incisor pulp tissues were removed.The pulp tis-sues were transfected with mirVana miRNA mimic miR-27a-5p or mirVana miRNA mimic Negative Control #1 (Thermo Fisher Scientific) using Lipofectamine RNAiMAX.Twentyfour hours after transfection, the pulp tissues were stimulated ex vivo with 200 ng/mL LPS for 4 h.Then, the pulp tissues were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde at 4 • C overnight.They were embedded in O.C.T. Compound and cryosectioned at 10 µm for immunofluorescence analysis.

Reverse Transcription-Quantitative Polymerase Chain Reaction
The QuickGene-Mini80 nucleic acid isolation system (FUJIFILM Wako Pure Chemical) was used to extract total RNA.Reverse transcriptase (RevertAid H Minus Reverse Transcriptase, Thermo Fisher Scientific) and PrimeScript™ RT Master Mix (Takara Bio, Kusatsu, Japan) were used to synthesize cDNA and perform RT-qPCR.mRNA detection was performed using a real-time PCR detection system (CFX 96, Bio-Rad, Hercules, CA, USA).For miR-27a-5p, total RNA was extracted with a mirVana miRNA Isolation Kit (Thermo Fisher Scientific), and cDNA was synthesized using RT primers specific for miR-27a-5p and U6 in TaqMan microRNA Assays (Thermo Fisher Scientific) using a microRNA Reverse Transcription Kit (Thermo Fisher Scientific).For real-time PCR, the CFX 96 was used with a TaqMan Universal Master Mix II, no UNG (Thermo Fisher Scientific).The formula 2 −∆∆Ct was used to calculate relative gene expression with ACTB or U6 as internal controls.Table 1 shows the primer sequences.

Cytometric Bead Array
The culture supernatant of hDPCs was collected for analysis.The Human Inflammation Standard (BD Biosciences, Franklin Lakes, NJ, USA) was reconstituted using Assay Diluent (BD Biosciences) and then mixed with Human Inflammation Capture Bead suspension for IL-6, IL-8, and MCP1 (BD Biosciences) diluted in Bead Capture Diluent (BD Biosciences).Cytokine detection was performed using phycoerythrin-conjugated antibodies.Quantification was based on a standard curve plot.Fluorescence from the phycoerythrin-conjugated antibodies was detected with a FACS Canto II flow cytometer (BD Biosciences), following the manufacturer's protocol.Data analysis was performed using FCAP Array Software v3.0 (BD Biosciences).
For the luciferase assay with the TAB1 3 ′ -UTR reporter vector, insertion of synthesized TAB1 3 ′ -UTRs containing wildtype or mutated hsa-miR-27a-5p target sequences (400 bp each; Eurofins Genomics, Ebersberg, Germany) was carried out in XhoI and HindIII sites of the pMIR-REPORT vector (Thermo Fisher Scientific).Using Lipofectamine 3000, these vectors were co-transfected into hDPCs with the miR-27a-5p mimic or NC.The cells were lysed using luciferase cell culture lysis reagent.A luminometer (Luminescence PSN, Atto) and luciferase assay equipment (Promega) were used to measure luciferase activity.

Statistical Analysis
Each experiment was performed at least three times.Results are expressed as the mean and standard deviation.One-way analysis of variance followed by the post-hoc Tukey-Kramer test or Bonferroni's test was employed for multiple comparisons.Student's t-test was used to compare the results of two groups.p < 0.05 was considered statistically significant.

Conclusions
Upregulation of miR-27a-5p and proinflammatory cytokines IL-6, IL-8, and MCP1 was induced in LPS-stimulated hDPCs, which was mediated through an NF-κB-dependent mechanism.Moreover, miR-27a-5p suppressed the expression of proinflammatory cytokines via NF-κB signaling by directly targeting TAB1, IRAK4, RELA, and FSTL1 in hDPCs stimulated with LPS.MiR-27a-5p-binding sequences in the TAB1 3 ′ -UTR were determined, and knockdown of TAB1 induced downregulation of proinflammatory mediator expression and NF-κB activity.Comparable observations were replicated in LPS-stimulated rat pulp tissues ex vivo.These results contribute to the understanding of the putative negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-κB signaling pathway, on pulp inflammation.

Table 1 .
Sequences for primers used in RT-qPCR.