Inhibition of circular RNA circ_0138959 alleviates pyroptosis of human gingival fibroblasts via the microRNA-527/caspase-5 axis.

Circular RNA (circRNA) plays a regulatory role in periodontitis. This study explored whether circ_0138959 affected lipopolysaccharide (LPS)-induced pyroptosis in human gingival fibroblasts (HGFs). The periodontal ligament (PDL) tissues and HGFs were derived from patients with periodontitis and healthy volunteers. HGFs treated with LPS were considered to mimic periodontitis in vitro. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the mRNA expression levels of circRNAs, miR-527, and caspase-5 (CASP5), and Western blotting assay was used to measure protein expression levels of caspase-1, caspase-4, and cleaved N-terminal gasdermin D (GSDMD-N). Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The concentration of lactate dehydrogenase (LDH), interleukin (IL)-1β, and IL-18 and the pyroptosis rate were determined to evaluate pyroptosis. The interaction between miR-527 and circ_0138959 or CASP5 was verified by dual-luciferase reporter and RNA pull-down assays. Circ_0138959 expression was higher in the PDL tissues of patients with periodontitis than in the healthy group; likewise, circ_0138959 was also upregulated in LPS-treated HGFs. Suppressed circ_0138959 increased cell viability and decreased pyroptosis of HGFs induced by LPS. miR-527 was a target of circ_0138959, and inhibition of miR-527 contributed to the dysfunction of LPS-treated HGFs and reversed the protective effects of downregulated circ_0138959. Additionally, miR-527 targeted CASP5. Increased CASP5 abrogated the effects of overexpressed miR-527 on cell viability and pyroptosis of LPS-treated HGFs. Inhibition of circ_0138959 promoted cell viability and suppressed pyroptosis of HGFs via the miR-527/CASP5 axis. Therefore, knockdown of circ_0138959 may be a promising therapy for periodontitis.

Introduction

Periodontitis is a common chronic inflammatory oral disease, which causes alveolar bone resorption, root exposure, and tooth loosening and loss by disrupting the bone formation/resorption balance between osteoblasts and osteoclasts [1,2]. Periodontitis is the most common cause of tooth loss in adults [3].

Porphyromonas gingivalis-lipopolysaccharide (LPS) of gram-negative bacteria is regarded as an important pathogenic factor of bone loss in periodontitis [4,5]. LPS has high toxicity and antigenicity to periodontal tissue and can stimulate osteoclasts to produce bone resorption [6]. It has been reported that LPS can stimulate osteoblasts and osteoclasts to secrete various inflammatory factors leading to an inflammatory response [7]. LPS can also stimulate osteoblast production and secretion of inflammatory cytokines to activate osteoclasts and promote osteoclast maturation [8,9]. Although the destructive effect of LPS on bone tissue has been widely recognized, the specific pathogenic mechanism remains unclear.

Circular RNA (circRNA) is a subset of non-coding RNA (ncRNA), which has recently emerged as a new regulator of gene expression [10-12]. circRNA functions as a competing endogenous RNA (ceRNA) by sponging microRNAs (miRNAs) in various diseases [13-15]. Dysregulated circRNAs have also been identified in periodontitis, suggesting that they have regulatory effects [16]. For example, Li J and Xie R identified four aberrant upregulated circRNAs (circ_0099630, circ_0138960, circ_0138959, and circ_0107783) in patients with periodontitis through sequencing technology analyses [16].

In this research, we investigated the cellular function and molecular mechanism of circ_0138959 in periodontitis. The ceRNA mechanism may be involved in the process of periodontitis.

Materials and methods

Periodontal ligament (PDL) tissue

The process of obtaining PDL tissue and clinicopathologic characteristics (Table 1) of study subjects was approved by The First People’s Hospital of Changzhou Ethics Committee (2,015,015), and informed consent was obtained from all patients. The tissues from 30 patients with periodontitis with obvious alveolar bone absorption and obvious tooth loosening requiring tooth extraction and from 30 healthy volunteers who had teeth removed for orthodontic purposes were used in this study. After the root surface was rinsed repeatedly with phosphate buffered saline (PBS) solution (C0221A; Beyotime, Shanghai, China), one-third of the periodontal membrane tissue in the root was scraped and stored at – 80°C [17].

Table 1.

Clinicopathologic characteristics of study subjects

Clinicopathologic characteristics	n	Low	High	p-value
Age (years)				0.4906
< 65	17	7	10
≥ 65	13	7	6
Sex				0.2607
Male	14	5	9
Female	16	9	7
BMI (kg/m2)				0.3724
Normal,18.5–24.99	12	6	6
Overweight,25–29.99	10	3	7
Obesity I, 30–34.99	8	5	3
Order of severity				0.0190*
slight	7	6	1
medium	12	6	6
severe	11	2	9
Grade of periodontitis				0.0069*
Level 0	4	3	1
Level 1	15	10	5
Level 2	11	1	10

Level 0, individuals with a healthy periodontium and up to one proximal site with loss of attachment ≥ 3 mm; Level 1,presence of proximal attachment and loss ≥ 3 mm in ≥ 2 nonadjacent teeth; and Level 2, presence of proximal attachment loss ≥ 5 mm in ≥ 30% of teeth.

Human gingival fibroblasts (HGFs)

Fresh healthy gingival tissue isolated from healthy volunteers was fully rinsed with PBS solution containing 5% double antibody (C0221G; Beyotime, Shanghai, China) to remove any blood clots and residual oral microorganisms on the surface [18], and the primary culture was conducted using a conventional tissue block method. When 70%–80% of the bottle was covered, 0.25% trypsin (SM-2001-C; Sigma-Aldrich, Missouri, USA) was used for 1:2 subculture, and the third generation cells were taken for the experiment. Isolated HGFs were then treated with 10 μg/mL LPS derived from Porphyromonas gingivalis (ST1470; Beyotime, Shanghai, China) to establish periodontitis model cells.

Lentivirus construction and cell transfection

The short hairpin RNA (shRNA) lentivirus of circ_0138959, negative control (sh-nc) lentivirus, overexpressed caspase-5 (CASP5) lentiviral vector (len-CASP5), and negative control (len-nc) were constructed by GenePharma Co., Ltd. (Jiangsu, China). According to previous studies [19,20], the HGFs of each group were transfected with the specified vector and/or sequence using Lipofectamine 3000 (Invitrogen, California, USA) reagent for 48 h when 60%–70% convergence was achieved.

qRT-PCR

PDL tissues and HGF samples of each group were selected, and total RNA was extracted in three parallel copies. After determining the RNA concentration, cDNA was synthesized according to the instructions of the reverse transcription kit (18,064,071; SuperScript ™II, Thermo Fisher, Shanghai, China). Real-time quantitative RT-PCR was conducted according to the PCR kit instructions (11,746,500; SuperScript™ III Platinum™ SYBR™ Green; Invitrogen, Shanghai, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the reference gene to detect the mRNA expression of circ_0138959 and CASP5. U6 was used as an internal reference to detect the expression of miR-527 [21]. The primer sequences are shown in Table 1. The PCR procedure was conducted at 95°C for 120 s and 95°C for 20 s, 60°C for 25 s, and 72°C for 20 s, for 40 cycles. The data were analyzed using the 2−ΔΔCt relative expression method. All experiments were repeated thrice.

Actinomycin D and Ribonuclease R (RNase R) treatment

RNase R (Epicenter, WI, USA) and actinomycin D (2 mg/mL, Sigma-Aldrich, Missouri, USA) were used to test the stability of circ_0138959 measured by qRT-PCR. RNase R from Escherichia coli is a magnesia-dependent 3ʹ→5ʹ exonuclease capable of digesting essentially all linear RNA, but it does not easily digest lasso or circRNA structures [22]. Total RNA was incubated for 1 h at 37°C with 3 U/μg of RNase R.

Cell counting kit-8 (CCK-8)

The HGFs in each group were inoculated into 96-well plates (2,000 cells/100 μL/well), and 10 μL CCK-8 solution (C0038; Beyotime, Shanghai, China) was added to each well and incubated in a cell incubator (37°C, 5% CO2) for 2 h [23]. Finally, the absorbance A value was detected at 450 nm wavelength. This was repeated for each group of samples five times, and the results were averaged.

Lactate dehydrogenase (LDH) measurement

An LDH Cytotoxicity Assay Kit (C0016; Beyotime, Shanghai, China) was used to measure the LDH levels in the HGFs of each group following the manufacturer’s instructions [24].

Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to detect the secretion of IL-1β and IL-18 by the HGFs of each group using a IL-1β ELISA kit (PI305; Beyotime, Shanghai, China) and a IL-18 ELISA kit (PI558; Beyotime, Shanghai, China) following the manufacturer’s instructions [25].

Flow cytometry assay

Cell pyroptosis was detected using a FAM-FLICA® Caspase-1 (YVAD) Assay Kit (biopike, Minnesota, USA). The HGFs in each group were scraped off and washed with PBS (C0221A; Beyotime, Shanghai, China) twice, before resuspending in caspase-1 staining solution (1:30). After 1 h of incubation in a 5% CO2 incubator at 37°C, PI staining solution (1:100) was added to resuspend the cells [26].

Western blotting assay

Protein expressions of caspase-1, Capase-4, and cleaved N-terminal gasdermin D (GSDMD-N) were measured by Western blotting [26]. Proteins were extracted from cells and tissues by RIPA (Sigma-Aldrich, Missouri, USA) reagents and were detected using the BCA method (P0012; Beyotime, Shanghai, China). Next, 30 μg total protein from each well was separated by 10% SDS-PAGE electrophoresis (P0690; Beyotime, Shanghai, China) and transferred to a PVDF membrane (FFP32; Beyotime, Shanghai, China). The membranes were blocked with 5% defatted milk for 1 h. Then, primary antibodies, including anti-caspase-1 (ab207802, 1/1000), anti-Capase-4 (ab238124, 1/1000), and anti-GSDMD-N (ab215203, 1/1000), were added (Abcam, California, USA). The next day, HRP labeled secondary antibody (ab7090, Abcam, California, USA) was added after the membrane was washed with PBS, and the membrane was incubated at room temperature for 1 h. The membranes were developed using ECL reagents with GAPDH as control.

Verification of relationships between mRNA and miRNA

The binding sites between miR-527 and circ_0138959 or CASP5 have been predicted through two online datasets, Starbase (http://starbase.sysu.edu.cn) and TargetScan 7.2 (http://www.targetscan.org) [27]. The wild type (wt) and mutant type (mut) 3ʹ-UTR region of circ_0138959 and the CASP5 luciferase reporter vectors were designed and synthesized by RiboBio (Guangzhou, China). Dual-luciferase reporter and RNA pull-down assays were conducted to verify the interactions between miR-527 and circ_0138959 or CASP5. For dual-luciferase reporter assay (16,186; Thermo Fisher, USA), circ_0138959 3ʹ-UTR (wt and mut) or CASP5 3ʹ-UTR (wt or mut) vectors containing binding sites with miR-527 were inserted into the firefly luciferase gene and then co-transfected into the HGFs of each group using Lipofectamine 3000 (L3000075; Thermo Fisher, USA).

For RNA pull-down assay, 500 μg streptavidin magnetic beads were combined with 200 pmol biotin-labeled miR-527 mimic and added to the total RNA extracted from the HGFs of each group. The pulled RNA complex was collected following the addition of eluting buffer at room temperature and incubation for 30 min. Finally, the circ_0138959 and CASP5 levels were quantitatively analyzed by qRT-PCR.

Statistical analysis

GraphPad Prism version 8.3 (GraphPad Software, CA, USA) was used to analyze all data, which are presented as mean ± standard deviation (SD). Student’s t-test (two groups) and one-way analysis of variance (ANOVA) followed by Tukey’s-test (multiple groups) were applied for statistical analysis. P-values < 0.05 were deemed statistically significant.

Results

Circ_0138959 is abnormally upregulated in periodontitis

Firstly, we detected expression of sevel circRNAs to identify the most aberrant expressed circRNA in periodontitis. Circ_0138959 was the most significantly increased circRNA in three pairs of PDL tissues from patients with periodontitis as well as healthy controls among four screened circRNAs (circ_0099630, circ_0138960, circ_0138959, and circ_0107783) [16] (Figure 1(a)). The circ_0138959 was further verified to be aberrantly upregulated in larger sample capacity periodontitis PDL tissues as well as LPS-treated HGFs (Figure 1(b,c)). In addition, the high expression of circ_0138959 was significantly associated with the severity and high grade of periodontitis (Table 1). The results of RNase R treatment indicated that the expression level of long non-coding (lnc)_0138959 was substantially decreased compared to that of circ_0138959 (Figure 1(d)). Circ_0138959 showed more stable mRNA expression compared to the linear group (Figure 1(e)).

Figure 1.

Circ_0138959 is abnormally upregulated in periodontitis.

Knockdown of circ_0138959 suppresses pyroptosis of HGFs

Then, circ_0138959 was inhibited to confirm the role in HGFs. Circ_0138959 expression was obviously decreased in the sh-circ_0138959 group (Figure 2(a)). Inhibition of circ_0138959 markedly restored cell viability, inhibited the secretion of LDH, IL-1β, and IL-18, and alleviated the pyroptosis rate of LPS-treated HGFs (Figure 2(b-f)). Furthermore, sh-circ_0138959 suppressed the protein levels of pyroptosis-related proteins, including caspase-1, caspase-4, and GSDMD-N in LPS-treated HGFs (Figure 2(g)).

Figure 2.

Knockdown of circ_0138959 suppresses pyroptosis of HGFs.

Circ_0138959 sponges miR-527 in HGFs

Afterward, interaction relationship between circ_0138959 and miR-527 were verified. The sequence alignment of miR-527 with the putative binding sites within the wt or mut regions of circ_0138959 is indicated in Figure 3(a). The luciferase activity of HGFs co-transfected with agomiR-527 and wt-circ_0138959 was dramatically decreased, while there were no significant changes in the mut group (Figure 3(b)). The results of RNA pull-down experiments showed that the expression level of circ_0138959 in the miR-527 probe group that was biotin-labeled (biotin-miR-527) was markedly higher than that in the control group (biotin-nc), which also confirmed the binding relationship between circ_0138959 and miR-527 (Figure 3(c)). Moreover, miR-527 was upregulated by suppressing circ_0138959 in HGFs (Figure 3(d)), and miR-527 expression levels were lower in PDL tissues of patients with periodontitis and LPS-treated HGFs compared to the healthy group (Figure 3(e,f)).

Figure 3.

Circ_0138959 sponges miR-527 in HGFs.

Upregulation of miR-527 reversed the effects of circ_0138959 in HGFs

Then, we investigated the role of miR-527 in HGFs by up-regulating or down-regulating miRNA expression levels. As indicated in Figure 4(a), the expression of miR-527 was dramatically decreased by miR-527 antagomir and increased by miR-527 agomir, revealing successful regulation of miR-527 expression by transfection. The antagomiR-527 notably inhibited cell viability, increased the concentrations of LDH, IL-1β, and IL-18, and promoted the pyroptosis rate of LPS-treated HGFs transfected with sh-circ_0138959 (Figure 4(b-f)). Furthermore, antagomiR-527 elevated the protein levels of caspase-1, caspase-4, and GSDMD-N in LPS-treated HGFs induced by sh-circ_0138959 (Figure 4(g)).

Figure 4.

Upregulation of miR-527 reversed the effects of circ_0138959 in HGFs.

CASP5 is a downstream target gene of miR-527 in HGFs

Then, interaction relationship between CASP5 and miR-527 were verified. The predicted binding sites between CASP5 and miR-527 are shown in Figure 5(a). Dual luciferase reporter and RNA pull-down assays verified the interaction between CASP5 and miR-527 (Figure 5(b,c)). The mRNA expression of CASP5 was notably suppressed by sh-circ_0138959 and elevated by antagomiR-527 in HGFs (Figure 5(c)). Additionally, CASP5 was dramatically upregulated in the PDL tissues of patients with periodontitis and LPS-treated HGFs compared to the healthy group (Figure 5(e,f)).

Figure 5.

CASP5 is a downstream target gene of miR-527 in HGFs.

Overexpressed CASP5 alleviates the effects of agomiR-527

Finally, we investigated the role of CASP5 in HGFs by up-regulating its expression levels. CASP5 expression was obviously increased in HGFs following transfection with len-CASP5 vectors (Figure 6(a)). Moreover, len-CASP5 suppressed cell viability of LPS-treated HGFs induced by agomiR-527. Similarly, the secretion of LDH, IL-1β, and IL-18 and the pyroptosis rate of LPS-treated HGFs transfected with antagomiR-527 were promoted by len-CASP5 (Figure 6(b-f)). Furthermore, the protein levels of caspase-1, caspase-4, and GSDMD-N in LPS-treated HGFs co-transfected with induced antagomiR-527 and len-CASP5 were dramatically higher than those in the LPS+antagomiR-527+ len-nc group (Figure 6(g)).

Figure 6.

Overexpressed CASP5 alleviates the effects of agomiR-527.

Discussion

In this study, we demonstrated that circ_0138959 contributed to the dysfunction of HGFs by competing with CASP5 to bind with miR-527 in periodontitis in vitro. Functionally, the circ_0138959/miR-527/CASP5 axis regulates the viability and pyroptosis of LPS-treated HGFs. Knockdown of circ_0138959 restores the function of HGFs and represents a promising treatment for periodontitis.

Many endogenous, abundant, and conserved circRNAs have been found in mammalian cells with the development of RNA sequencing technology and bioinformatics [10]. As circRNAs are not easily degraded, circRNAs can also be used for disease diagnosis and analysis of biomarkers [10,28]. For instance, Du W et al. recommended circ_0085289 as a biomarker for periodontitis treatment by analyzing the regulatory roles of circ_0085289 in LPS-treated PDLC [5]. Additionally, circRNAs can act as ceRNAs in organisms, thereby inhibiting the activity of miRNAs and regulating the expression of target genes [29-31]. Li et al. found that circRNA CDR1 promoted the osteogenic differentiation of PDLSCs via the miR-7/GDF5/SMAD axis [32]. Wang et al. found that circRNA expression changed during the osteogenic differentiation of PDLSCs induced by mechanical force [33]. These studies indicate that circRNAs play important regulatory roles in periodontitis.

Li J and Xie R identified four aberrant upregulated circRNAs (circ_0099630, circ_0138960, circ_0138959, and circ_0107783) in patients with periodontitis through sequencing technology analyses [16]. Our data suggested that circ_0138959 was the most aberrant dysregulated among the four circRNAs in the PDL tissues of patients with periodontitis. Furthermore, LPS-treated HGFs were established to mimic periodontitis in vitro, and the expression of circ_0138959 was verified to be abnormally upregulated in LPS-treated HGFs, demonstrating that circ_0138959 may be a key regulator in periodontitis.

Pyroptosis is a type of pro-inflammatory programmed cell death, between apoptosis and necrosis, which is characterized by DNA damage, membrane perforation, and the release of IL-1β and IL-18 [34]. Increasing evidence has demonstrated that inflammatory caspases, including caspase-1, contribute to pyrotosis through different signaling pathways [35]. Recent studies have shown that Porphyromonas gingivalis (PG), the main cause of periodontitis, can induce pyroptosis by its virulence factors [1]. The outer membrane vesicles on the surface of PG can invade host cells through various mechanisms and transfer the virulence factors contained in the vesicles to the host tissues [36,37]. The outer membrane vesicles are rich in the main virulence factors of pathogens, such as LPS [38]. LPS can increase the expression of GSDMD-N by activating the inflammasome and caspase-1 and mediate the pyroptosis of classical and non-classical pathways [39,40]. As reported previously, pyroptosis of HGFs and macrophages could accelerate the process of periodontitis [41,42]. Therefore, pyroptosis may play a vital role in periodontitis. Our data revealed that cell viability was restored and pyroptosis was suppressed when circ_0138959 was suppressed in LPS-treated HGFs.

Growing evidence suggests that circRNA and miRNA suppress each other as ceRNAs and form an accurate regulatory network to regulate target genes of miRNA [43,44]. Our data suggested that miR-527 is a target miRNA of circ_0138959 and could be upregulated by inhibition of circ_0138959. miR-527 is a newly discovered tumor suppressor in non-small cell lung cancer (NSCLC), bladder cancer, and glioma [45-47]. However, there are almost no reports of miR-527 in periodontitis. Our results indicated that miR-527 was suppressed in the PDL tissues of patients with periodontitis as well as LPS-treated HGFs, suggesting that miR-527 plays a regulatory role in periodontitis. We confirmed this hypothesis by functional validation. Downregulation of miR-527 partly abrogated the effects of sh-circ_0138959 on cell viability and pyroptosis of LPS-treated HGFs, demonstrating that miR-527 may help to restore the function of HGFs to alleviate periodontitis. These findings agreed with those of Lian J et al., who illustrated that miR-335-5p functioned as a bone loss suppressor to prevent periodontitis [48].

Furthermore, CASP5 was predicted and verified to be a downstream gene of miR-527, and CASP5 expression was positively regulated by circ_0138959 and negatively regulated by miR-527 in HGFs in this study. Caspase belongs to a family of cysteine proteases that are involved in programmed cell death, such as necrosis, pyroptosis, and apoptosis, as well as inflammation [49]. Caspase-1, caspase-4, and caspase-5 are associated with inflammation and can activate pyroptosis [50]. Our data also suggested that overexpression of the CASP5 gene not only activated pyroptosis but also suppressed cell viability to alleviate the functions of agomiR-527 in LPS-treated HGFs.

Conclusion

Our research suggested that circ_0138959 acted as a ceRNA to contribute to the dysfunction of HGFs via the miR-527/CASP5 axis in periodontitis in vitro. Therefore, knockdown of circ_0138959 may be an alternative treatment for periodontitis.