Cuiwei Liang,Wenmiao Li,Qian Huang,Qitao Wen
CircFKBP5 Suppresses Apoptosis and Inflammation and Promotes Osteogenic Differentiation
CircFKBP5,Inflammation,hDPSCs,Osteogenic differentiation,Pulpitis
Objectives Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell possessing self-renewal and multilineage differentiation capabilities. The dysfunction of DPSCs is related to the pathologic process of pulpitis. The participation of circular RNAs (circRNAs) in DPSC differentiation has been identified. This work focussed on exploring the functions and mechanism of circFKBP5 in DPSC dysfunction evoked by lipopolysaccharide (LPS). Materials and methods The viability and apoptosis of human DPSCs (hDPSCs) were determined using Cell Counting Kit-8 assay and flow cytometry. Inflammation was analysed by measuring the release of inflammatory cytokines. The osteogenic differentiation of hDPSCs was investigated by performing alkaline phosphatase (ALP) staining and alizarin red S staining and detecting the changes of ALP and runt-related transcription factor 2 (RUNX2) proteins. The dual-luciferase reporter, RNA immunoprecipitation (RIP), and pull-down assays were used to confirm the binding between miR-708-5p and circFKBP5 or G-protein-coupled receptor (GPCR)–kinase interacting protein 2 (GIT2). Results CircFKBP5 expression was decreased in hDPSCs and, functionally, reexpression of circFKBP5 attenuated LPS-induced apoptosis, inflammation, and inhibition of proliferation ability and osteogenic differentiation in hDPSCs. Mechanistically, circFKBP5 acted as a sponge for miR-708-5p, which was verified to target GIT2. LPS induced miR-708-5p expression in hDPSCs, and knockdown of miR-708-5p protected against LPS-evoked hDPSC dysfunction. Besides, GIT2 expression was decreased in hDPSCs after LPS treatment. Rescue experiments showed that GIT2 could mediate the protective functions of circFKBP5 on hDPSCs under LPS treatment. Conclusions CircFKBP5 could protect against LPS-induced apoptosis, inflammation, and osteogenic differentiation inhibition in hDPSCs via the miR-708-5p/GIT2 axis.
CircFKBP5 Suppresses Apoptosis and Inflammation and Promotes Osteogenic Differentiation

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell possessing self-renewal and multilineage differentiation capabilities. The dysfunction of DPSCs is related to the pathologic process of pulpitis. The participation of circular RNAs (circRNAs) in DPSC differentiation has been identified. This work focussed on exploring the functions and mechanism of circFKBP5 in DPSC dysfunction evoked by lipopolysaccharide (LPS).

The viability and apoptosis of human DPSCs (hDPSCs) were determined using Cell Counting Kit-8 assay and flow cytometry. Inflammation was analysed by measuring the release of inflammatory cytokines. The osteogenic differentiation of hDPSCs was investigated by performing alkaline phosphatase (ALP) staining and alizarin red S staining and detecting the changes of ALP and runt-related transcription factor 2 (RUNX2) proteins. The dual-luciferase reporter, RNA immunoprecipitation (RIP), and pull-down assays were used to confirm the binding between miR-708-5p and circFKBP5 or G-protein-coupled receptor (GPCR)–kinase interacting protein 2 (GIT2).

CircFKBP5 expression was decreased in hDPSCs and, functionally, reexpression of circFKBP5 attenuated LPS-induced apoptosis, inflammation, and inhibition of proliferation ability and osteogenic differentiation in hDPSCs. Mechanistically, circFKBP5 acted as a sponge for miR-708-5p, which was verified to target GIT2. LPS induced miR-708-5p expression in hDPSCs, and knockdown of miR-708-5p protected against LPS-evoked hDPSC dysfunction. Besides, GIT2 expression was decreased in hDPSCs after LPS treatment. Rescue experiments showed that GIT2 could mediate the protective functions of circFKBP5 on hDPSCs under LPS treatment.

CircFKBP5 could protect against LPS-induced apoptosis, inflammation, and osteogenic differentiation inhibition in hDPSCs via the miR-708-5p/GIT2 axis.

Pulpitis is an inflammatory reaction of the pulp typically caused by an opportunistic infection of commensal oral microorganisms and is clinically categorised as reversible and irreversible pulpitis. Dental pulp stem cells (DPSCs) are one type of mesenchymal stem cell (MSC) possessing self-renewal and multilineage differentiation capabilities. They are able to differentiate into osteoblasts, adipocytes, odontoblasts, and chondrogenic lineages, which play significant roles in tooth development and injury repair. In the pulpitis process, it has been revealed that pro-inflammatory cytokines induced by bacterial irritants can affect the survival and differentiation of DPSCs. Thus, in-depth investigations on the mechanism of DPSC dysfunction is of great significance for pulpitis management. Circular RNAs (circRNAs) are a type of endogenous molecules without 5’ cap and 3’ poly (A) tail. Due to the round structure, circRNAs fail to be degraded by exonucleases and show higher stability compared with other linear RNAs. Emerging data have shown the importance of circRNAs in diverse pathologic and physiologic processes associated with cell apoptosis, metabolism, and inflammation.7, 8, 9 In addition, emerging evidence indicates that circRNAs are implicated in the osteogenic diferentiation of stem cells by serving as microRNA (miRNA) sponges. For example, circRNA-23525 promoted osteogenic processes by inducing osteogenesis in adipose-derived MSCs via upregulating RUNX2 expression through miR-30a-3p. circRNA_33287 reexpression could increase osteogenic differentiation by miR-214-3p/RUNX3 in maxillary sinus membrane stem cells. Further, Ji et al showed that hsa_circ_0026827 facilitated osteoblast differentiation and autophagy in human DPSCs (hDPSCs) by extricating miR-188-3p–mediated inactivation of the beclin1 and RUNX1 pathway. In addition, circFKBP5 (ID: hsa_circ_0001599 or hsa_circRNA_104101) knockdown in hDPSCs suppressed odontogenic differentiation. However, the action and mechanism of circFKBP5 on hDPSC dysfunction remain unclear. G-protein-coupled receptor (GPCR)-kinase interacting protein 2 (GIT2) is a member of GIT family of GPCR scaffolding proteins that can modulate various cellular functions, such as lipid metabolism, cell apoptosis, and cytoskeletal dynamics via interacting with a large number of proteins., Besides, GIT2 has effects on adaptive and innate immunity. Bao et al showed that GIT2 was elevated during odontogenic differentiation, and GIT2 silencing could suppress odontogenic differentiation via the MALAT1 lncRNA/miR-140-5p/GIT2 axis in hDPSCs. However, whether GIT2 mediates the action of circFKBP5 remains unclear. Lipopolysaccharide (LPS), the major molecular component of the cell walls of Gram-negative bacteria, is a prime toxic factor inducing bacterial-evoked immune response and has been commonly used to evoke a model of inflammation in vitro., Herein, we used LPS to mimic the inflammatory condition of pulpitis and then investigated the function of circFKBP5 on hDPSC survival, inflammation, and osteogenic differentiation. In addition, a competing endogenous RNA (ceRNA) network was constructed to elucidate the potential mechanism/s of circFKBP5 and hDPSCs interactions.

The hDPSCs from human dental pulp tissue were obtained from Lonza (Basel, Switzerland). hDPSCs were recovered with an AccuVital cell recovery kit (AccuRef Scientific) and then cultivated in the α-modification of Eagle's medium (α-MEM, Gibco) plus 10% fetal bovine serum (FBS; Gibco) in a 5% CO2 incubator at 37 °C. To induce osteogenic differentiation, hDPSCs were grown in the α-MEM harbouring 20% FBS, 100 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate (β-GP), and 50 mg/mL ascorbic acid (osteogenic medium) with 5% CO2 at 37 °C for 14 days. For LPS treatment, hDPSCs were exposed to 100 ng/mL LPS (Sigma-Aldrich) for 24 hours before collection.

The PARIS™ Kit (Invitrogen) was utilised to separate RNAs from nucleus and cytoplasm of hDPSCs under the manufacturer's recommended conditions. Total RNAs were isolated by using TRIzol reagent (Invitrogen) and quantified with the NanoDrop ND-1000. The treatment of RNase R (3 U/μg, Geneseed) was performed with 3 μg of total RNA at 37 °C for 30 minutes. Total RNA was reverse-transcripted into complementary DNA using a reverse transcription system kit (Takara), and qRT-PCR analysis was subsequently conducted with SYBR premix ExTaq (Takara). The relative expression normalised to GAPDH or U6 was assayed by 2−ΔΔCt method. The primer sequences are listed as follows: circFKBP5, Forward GCATTATCCGGAGAACCAAA, Reverse TGGGGCTTTCTTCATTGTTC; FKBP5, Forward CGGCGACAGGTTCTCTACTT, Reverse ATCGGCGTTTCCTCACCATT; miR-642a-5p, Forward GTATGAGTCCCTCTCCAAATGTGT, Reverse CTCAACTGGTGTCGTGGA; miR-561-5p, Forward GTATGAATCAAGGATCTTAAACT, Reverse CTCAACTGGTGTCGTGGA; miR-28-5p, Forward GTATGAAAGGAGCTCACAGTCTA. Reverse CTCAACTGGTGTCGTGGA; miR-708-5p, Forward GTATGAAAGGAGCTTACAATCTAGC, Reverse CTCAACTGGTGTCGTGGA; miR-3139, Forward GTATGATAGGAGCTCAACAGATGC, Reverse CTCAACTGGTGTCGTGGA; miR-545-5p, Forward GTATGATCAGTAAATGTTTATTAG, Reverse CTCAACTGGTGTCGTGGA; miR-1271-5p, Forward GTATGACTTGGCACCTAGCAAGCA. Reverse CTCAACTGGTGTCGTGGA’ GIT2, Forward ACCTCAAATGGCAGACAGCA, Reverse CGTCTCTCGCCTGTCAACTT; GAPDH, Forward AGAAGGCTGGGGCTCATTTG, Reverse AGGGGCCATCCACAGTCTTC; U6, Forward CGCTTCACGAATTTGCGTGTCAT, Reverse GCTTCGGCAGCACATATACTAAAAT.

pCD5-ciR circFKBP5 overexpression plasmids (circ_0018429), siRNAs targeting GIT2 (si-GIT2), and miR-708-5p mimics (miR-708-5p) or inhibitors (in-miR-708-5p) were constructed by GenePharma with empty pCD5-ciR plasmids (vector), nontarget siRNAs (si-NC), miR-NC, or in-miR-NC as contrast. Then all the aforementioned plasmids or oligonucleotides alone or in combination were individually transduced into hDPSCs using Lipofectamine 3000 (Invitrogen).

hDPSCs were seeded into the 96-well plates (5 × 103 cells/well) ovenight and then reactedwith 10 μL CCK-8 solution (Solarbio) at 37 °C for 2 hours’ incubation. Later, cell viability was represented by the absorbance at 450 nm assayed by a microplate reader.

hDPSCs in different groups were resuspended with phosphate buffer saline (PBS) solution (1 × 106/mL). Further, 1 mL of cell suspension was dyed with staining buffer harbouring with 5 μL fluorescein isothiocyanate (FITC) and 5 μL propidium iodide (BestBio) for 15 min under darkness. Finally, apoptosis analysis under the flow cytometry (Applied Biosystems) was conducted within 1 hour.

hDPSCs were lysed using RIPA buffer (Beyotime), and the concentration of extracts was quantified using the bicinchoninic acid (BCA) method. Samples (40 μg of protein) were fractionated by SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore), followed by sealing with 5% nonfat milk for 1 hour. Thereafter, the membranes were incubated with primary antibodies at 4 °C for 24 hours and then reacted with secondary antibodies conjugated by HRP for 2 hours at 37 °C. Finally, an ECL reagent (Beyotime) was applied to quantify protein bands. The primary antibodies included B-cell lymphoma-2 (Bcl-2; ab59348), Cleaved Caspase-3 (c-caspase3; ab184787), Bcl2-Associated X (Bax; ab32503), runt-related transcription factor 2 (RUNX2; ab236639), alkaline phosphatase (ALP; ab83259), β-actin (ab8227; 1:1000) obtained from Abcam, and GIT2 (PA5-78301; Thermo Fisher Scientific).

The culture supernatant was collected from indicated hDPSCs by centrifugation at 2500 g for 10 minutes. Then, 20 μL supernatant was incubated with 50 μL antibody at 37 °C for 2 hours following the instructions of the corresponding ELISA kits (Abcam), and levels of interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) were calculated by reading the optical density values at 450 and 570 nm.

hDPSCs were cultured in a 12-well plate with osteogenic medium for 2 weeks. Cells were fixed with 4% paraformaldehyde (Solarbio) for 10 minutes, and then ALP staining was conducted according to the NBT/BCIP staining kit (Cwbiotech). Then ALP activity was assessed by an ALP detection kit (Beyotime), and ALP staining was observed using an inverted microscope at 100× magnification (Bio-Rad).

hDPSCs were seeded into a 12-well plate and cultured with osteogenic medium for 21 days. After being fixed with 4% formaldehyde (Solarbio) for 30 minutes, cells were stained with 2% ARS solution (Solarbio) at 37 °C for 10 minutes, followed by eluting with hexadecylpyridinium chloride (100 mM, Sigma-Aldrich) for 1 hour. Later, the absorbance was detected at 562 nm by a microplate reader (Bio-Rad).

Biotin-labelled circFKBP5 probe (circFKBP5 probe) and the control oligonucleotide probes (Oligo probe), as well as biotin-labelled miR-708-5p probe (Bio miR-708-5p) and the control probe (Bio-miR-NC) were synthesised by Geneseed. hDPSCs were lysed , the lysate was then incubated with these constructed probes. Then streptavidin-coupled magnetic beads were reacted with the lysates to pull down the biotin-coupled RNA complex. Finally, the complex was purified by TRIzol and subjected to qRT-PCR.

The pmirGLO report luciferase vector (Promega) containing the wild-type (WT) fragments or site sequences mutation (MUT) of circFKBP5 or GIT2 3’UTR in the presumptive miR-708-5p complementary sequence were constructed, termed as circFKBP5 WT, circFKBP5 MUT, GIT2 3’UTR WT, and GIT2 3’UTR MUT. Then the recombinant reporter plasmids together with miR-708-5p mimic or miR-NC were co-transfected into hDPSCs. Finally, luciferase activity was explored after 48 hours.

At this stage, 200 μL lysates of hDPSCs isolated by RIP lysis buffer (Sigma-Aldrich) were incubated with magnetic beads embracing Argonaute-2 (Ago2) or Immunoglobulin G (IgG) antibodies at 4 °C for 4 hours based on the instruction of Magna RIP RNA-binding protein immunoprecipitation kit (Millipore). Immunoprecipitated RNA was incubated with proteinase K (Solarbio) and then purified and subjected to qRT-PCR analysis.

The numerical data are shown as, mean ± standard deviation (SD). The test and control comparisons were carried out using Student t test (2 groups) or analysis of variance with Tukey posttest (multiple groups). P < .05 was considered statistically significant. Unless otherwise indicated, “n” indicates the number of individual experiments.

hDPSCs were treated with 100 ng/mL LPS for 24 hours to mimic the inflammatory condition of pulpitis. The results of qRT-PCR showed that LPS treatment decreased circFKBP5 expression in hDPSCs (Figure 1A). circFKBP5 is produced by back-splicing of the exon 3-6 of FKBP5 gene on chr6: 35586872-35610620 (Figure 1B). RNase R treatment could rapidly degrade linear FKBP5 but not the circFKBP5 in hDPSCs (Figure 1C). Besides that, the subcellular localisation analysis suggested that circFKBP5 was mainly distributed in the cytoplasm of hDPSCs (Figure 1D). In all, circFKBP5 is a relatively stable cytoplasmic transcript that was decreased by LPS treatment in DPSCs. Next, the detailed functions of expression circFKBP5 on hDPSCs was determined using gain-of-function analysis. Following the transfection of circFKBP5 plasmids or vector, qRT-PCR showed that circFKBP5 expression was significantly elevated after circFKBP5 plasmid introduction compared with vector transfection in hDPSCs (Figure 1E). Functionally, CCK-8 assay suggested that circFKBP5 overexpression attenuated LPS-induced inhibition of hDPSC viability (Figure 1F). In flow cytometry and western blotting analyses, LPS treatment induced apoptosis in hDPSCs, accompanied with the increases of Bax and C-caspase3 proteins as well as decrease of Bcl-2 protein, which were reversed by circFKBP5 overexpression (Figures 1G and 1H). Besides, the release of proinflammatory cytokines IL-1β and TNF-α caused by LPS was reduced after upregulation of circFKBP5 in hDPSCs (Figures 1I and 1J). During the osteoblastic differentiation, the ALP activity in the hDPSCs was significantly reduced by LPS treatment and subsequently increased upon circFKBP5 plasmid transfection; in addition, ARS staining showed that LPS treatment led to a lighter intensity of staining and reduced alizarin red positive nodules. These effects were rescued by circFKBP5 introduction, indicating that the efficiency of osteogenic differentiation is much greater under the circFKBP5 overexpression condition in hDPSCs (Figure 1K). Meanwhile, it was observed that circFKBP5 overexpression increased the levels of osteogenesis differentiation markers ALP and RUNX2 in LPS-stimulated hDPSCs (Figure 1L). Altogether, circFKBP5 could inhibit apoptosis and inflammation but promote osteogenic differentiation in hDPSCs.

Given that circFKBP5 was mainly distributed in the cytoplasm of hDPSCs, the underlying miRNAs interacting with circFKBP5 were investigated based on the prediction software circBank and starBase, and 7 miRNAs were selected from the overlap between the 2 databases (Figure 2A). Furthermore, RNA pull-down assay with a biotin-labelled circFKBP5 probe was conducted, and miR-708-5p with significantly enhanced fold-changes for circFKBP5 capture in hDPSCs was observed (Figure 2B). Thus, we speculated that circFKBP5 might target miR-708-5p. To validate this hypothesis, WT and mutant miR-708-5p targeted sites of circFKBP5 were constructed (Figure 2C). qRT-PCR analysis showed that miR-708-5p mimic markedly elevated miR-708-5p expression in hDPSCs (Figure 2D). Then, dual-luciferase reporter assay showed that miR-708-5p mimic could weaken the luciferase activity of WT circFKBP5 reporter vector, but not the mutated one, in hDPSCs (Figure 2E). In addition, RIP assay exhibited that miR-708-5p and circFKBP5 were effectively pulled down by anti-Ago2 antibody compared with the control IgG in hDPSCs (Figure 2F). Pull-down assay showed that circFKBP5 was notably captured by the biotin-labelled miR-708-5p probes compared with the control probes (Figure 2G). In addition, we found that LPS treatment increased miR-708-5p expression, whilst this elevation was reduced by circFKBP5 overexpression in hDPSCs (Figure 2H). Therefore, these data confirmed that circFKBP5 directly targeted miR-708-5p and suppressed its expression.

Next, the function of miR-708-5p on hDPSCs was explored. The interference efficiency of miR-708-5p inhibitor was first validated using qRT-PCR in hDPSCs (Figure 3A). After treatment with LPS, it was found that miR-708-5p inhibition reversed LPS-induced proliferation arrest (Figure 3B), apoptosis enhancement (Figures 3C and 3D), and reduction of IL-1β and TNF-α levels (Figures 3E and 3F) in hDPSCs. Besides that, miR-708-5p inhibition led to a promotion of osteogenic differentiation in LPS-stimulated hDPSCs (Figures 3G and 3H). Taken together, knockdown of miR-708-5p could repress apoptosis and inflammation and promote osteogenic differentiation in hDPSCs.

According to the prediction of starBase database, there was a conserved target site between miR-195-5p and GIT2 (Figure 4A). Then the dual-luciferase reporter assay was applied, and the results showed that miR-708-5p mimics could overtly reduce the luciferase activity of the WT group but not the mutant one in hDPSCs (Figure 4B). Furthermore, pull-down assay exhibited that GIT2 was significantly enriched in biotin-labelled miR-195-5p probes compared with the control probe (Figure 4C). Besides, we found that GIT2 expression was decreased by the miR-195-5p mimic and increased by the miR-195-5p inhibitor in hDPSCs (Figures 4D and 4E). These results confirmed that miR-195-5p suppressed GIT2 expression in a targeted way. Moreover, we elucidated whether there was a network amongst circFKBP5, miR-708-5p, and GIT2. As shown in Figures 4F and 4G, it was observed that circFKBP5 overexpression was accompanied with increased GIT2 expression, whilst miR-708-5p upregulation reversed circFKBP5-induced elevation of GIT2 in hDPSCs, indicating the circFKBP5/miR-708-5p/GIT2 feedback loop.

The knockdown efficiency of GIT2 siRNAs was first validated by western blotting with a decrease of GIT2 level in hDPSCs after si-GIT2 introduction (Figure 5A). Then hDPSCs were co-transfected with circFKBP5 and si-GIT2, followed by LPS treatment. Functionally, GIT2 knockdown abolished circFKBP5-evoked proliferation (Figure 5B), inhibition of apoptosis (Figures 5C and 5D), and reduction of IL-1β and TNF-α release levels (Figures 5E and 5F) in hDPSCs under LPS treatment. Meanwhile, the enhancement of ALP activity, increased alizarin red positive nodules, as well as elevation of ALP and RUNX2 proteins caused by circFKBP5 in LPS-stimulated hDPSCs were attenuated by GIT2 silencing (Figures 5G through 5I). Collectively, circFKBP5 could exert its functions via regulating GIT2.

Currently, root canal therapy is the traditional treatment for pulpitic teeth. It requires the removal of all pulp and the filling of the root canal, which increases the odds of extraction of the affected tooth and arrests the root development of immature permanent teeth. Previous findings have proposed that the restoration of DPSC functions confers a vital effect on the formation of pulp regeneration.21, 22, 23 Thus, targeting DPSC functioal rejuvenation may be a novel and promising therapeutic strategy for pulpitis. Currently, the participation of circRNAs in the differentiation of DPSCs has been identified., Based on the extant data,we investigated the biological features of circFKBP5 on DPSCs. We found that circFKBP5 expression was decreased by LPS treatment in DPSCs. Furthermore, overexpression of circFKBP5 in DPSCs reversed LPS-evoked apoptosis, inflammation, and osteogenic differentiation arrest. Thus, we have reason to believe that circFKBP5 may be involved in the pathogenesis of pulpitis. circRNAs in the cytoplasm can act as miRNA sponges to regulate gene expression at the posttranscriptional or transcriptional levels, as outlined in the ceRNAs hypothesis., Thus, we investigated whether circFKBP5 could regulate DPSC function via the miRNA/mRNA axis, and the potential targets of circFKBP5 The results suggested the existence of a circFKBP5/miR-708-5p/GIT2 feedback loop in hDPSCs. A number of studies have reported that the dysregulation of miRNAs is associated with the pathogenic behaviour of DPSCs. For example, Qiao et al showed that miR-224-5p suppressed DPSC apoptosis via decreasing Rac1 expression. Reexpression of miR-140-3p facilitated osteo-/dentinogenic differentiation of DPSCs by directly repressing KMT5B. In addition, Hao et al showed an increase of miR-708 in steroid-induced osteonecrosis of femoral head (ONFH) and glucocorticoid-treated MSCs, and miR-708 could inhibit osteogenic and adipogenesis differentiation in MSCs. Moreover, miR-708-5p suppressed osteoblast differentiation in bone marrow MSCs in an osteoporosis rat model. Surprisingly, this work demonstrated that miR-708-5p was increased by LPS treatment; additionally, inhibition of miR-708-5p protected hDPSCs from apoptosis, inflammation, and decreased osteogenic differentiation. In a previous study, the decreased bone volume and bone mineral density in both the trabecular and cortical compartments were observed in adult GIT2(−/−) mice; furthermore, impaired osteoblast differentiation was found in MSCs derived from adult GIT2(−/−) mice, underscoring the positive regulatory role of GIT2 in osteoclast and osteoblast function. In this report, a decreased GIT2 in hDPSCs after LPS treatment was observed, and further investigation exhibited that GIT2 silencing attenuated the protective effects of circFKBP5 on hDPSCs. Taken together, our work demonstrated that circFKBP5 could efficiently suppress apoptosis and inflammation and promote osteogenic differentiation in DPSCs under LPS treatment through the the miR-708-5p/GIT2 axis. Our findings provide a new insight into the action pattern of circFKBP5 in the pulpitis process and reveal a potential therapeutic target for patients with pulpitis.

None disclosed.