- Subjects
This protocol was approved by the Human Research Ethics Committee of Ninth People's Hospital. After reading information about the study and explaining the procedures, all participants signed a consent form. We used the spitting methods to collect saliva [15]. Briefly, we collected saliva in the morning (7:00 AM to 12:00 noon). The subjects sat upright in the dental chair and rinsed thoroughly with deionized water before collecting saliva according to instructions. Saliva is allowed to accumulate on the mouth floor. The subject spitted it out into the preweighed tube every 60 seconds. After collection of saliva, it was stored at 4℃ for up to 6 h, after which we held it at -80℃ until use.
- Salivary Exosomes Isolation and Detection
According to the manufacturer's instructions, we used Umibio® exosome isolation kits (Umibio, Cat. No: UR52121, China). In short, each sample was centrifuged at 3000 g 4 ℃ for 10 minutes and then at 10000 g 4 ℃ for 20 minutes to remove cells and debris. According to the manufacturer's instructions, we added the corresponding amount of reagent proportional to the volume of the starting sample. The mixture was well mixed, incubated at 4 ℃ for 2 hours, then centrifuged at 10000 g at 4 ℃ for 60 minutes to precipitate exosomes. The precipitate was resuspended with 1 x PBS and purified by exosome purification filter at 3000 g 4 ℃ for 10 minutes. The initial volume of exosome particles was 5 ml and the resuspension volume was 200 μ L. All exosomes were stored at - 80 ℃ immediately after extraction until further analysis. Zetaview PMX 110 (particle metrix, meerbusch, Germany) and transmission electron microscopy (TEM, jeol, jem-1230, TEM, Peabody, MA) were used to measure the exon size by nanoparticle tracking analysis (NTA).
- The miR-223 expression in Salivary Exosomes
We used the microRNA Reverse Transcription Kit ( EZBioscience, Cat. NO.: EZB-miRT2,USA) and TB GreenTM Premix Ex Taqtm (TaKaRa, Cat. NO.: RR420A, China) for the real time-PCR analyses. U6 was choosed as an internal reference for detecting miR-223-3p expression by the 2-ΔΔCt method.
- Target gene prediction and dual-luciferase reporter assay
Target gene prediction software, including miR-Base (http://www.mirbase.org/), TargetScan 4.2 (http://www.targetscan.org/), and PicTar (http://pictar.mdcberlin.de/) were used to predicted the miR-223-3p’s potential molecular targets. Among the potential targets, we focused on NLRP3 because it is highly expressed in inflammatory gingival tissue, as our previous studies have shown [16].
- Plasmid vectors construction and Dual-Luciferase Reporter Assays
The plasmids of wild type (wt)-NLRP3(clone ID: BK295, pmirGLO-NLRP3-3UTR-WT) and mutant (mut)-NLRP3-fused luciferase genes ((clone ID: BK296, pmirGLO-NLRP3-3UTR-MU) were constructed using conventional methods. One day before transfection, HEK293 cells in logarithmic growth phase were collected. After centrifugation and suspension, the cell density was adjusted, and the density of 1 x 105 cells at per hole was inoculated in 48 well plates. According to the manufacturer's instructions, all transfections were performed with Lipofectamine 3000 (Invitrogen). The cells were transfected with 1 μg pmirGLO luciferase expression vector containing 3'UTR of human NLRP3 (Promega) and 60 nM hsa-miR-223-3p mimics or blank control (Ribobio, China). After 48 hours of transfection, the Dual-Luciferase reporter analysis system (Promega) was used to measure the luciferase activities normalizing to Renilla luciferase activity. All the experiments were conducted three times independently, and the data came from three independent experiments.
- Integrated Bioinformatics Methods
The NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) database were used in Oct. 2019 using a single keyword "chronic periodontitis", and we found two relative datasets GSE10334 and GSE16134. Both microarray datasets were used GPL570 Platforms (HG-U133_Plus_2 Affymetrix Human Genome U133 Plus 2.0 Array). Both studies had obtained informed consent from all patients and been performed with the ethics committee's acquired approval in their institutions. The two gene expression datasets were further employed to identify the disease-associated pathways using the Gene Set Enrichment Analysis (GSEA; v4.0.2; http://software.broadinstitute.org/gsea/index.jsp). |NES| ≥1, NOM p-value ≤ 0.05, and FDR q value ≤ 0.25 were considered statistically significant.
- THP-1 culture and Differentiation
Human monocyte macrophage (THP-1) cell lines were obtained from ATCC and cultured in RPMI 1640 medium (Gibico, USA) containing 10% fetal bovine serum (Gibico, USA) and 2 mmol / L glutamine (Gibico, USA). THP-1 in logarithmic growth phase was collected for differentiation stimulation. Cells were collected and centrifugated. Cells were resuspended in a complete medium containing 200 nm phorbol-12-myristate-13-acetate (PMA, sigma Aldrich). The cell density was adjusted to 1 x 104 cells / ml, and the cells were inoculated for differentiation stimulation. Three days after inoculation, PMA treated THP-1 cells were gently washed with phosphate buffer (PBS) for three times, and replaced with fresh RPMI 1640 (10% FCS, 1% L-glutamine) containing Lipofectamine 3000 (Invitrogen life technologies, USA), and transiently transfected with miR-223-3p mimics, mir-223-3p inhibitor or control mimics (ribobio, China) for 48 hours. After transfection, THP-1-derived macrophages were treated with lipopolysaccharide (LPS) of P. gingivalis (1 μ g / ml) for 6 h, and real-time PCR was performed.
- Real time-PCR
THP-1-derived macrophages treated with P. gingivalis-LPS were collected and exracted RNA by Trizol reagent (Invitrogen). The PrimeScriptTM RT Kit (Shanghai Roche Pharmaceutical Co., Ltd., Shanghai, China) was used to prepare the cDNA. The expression levels of Caspase-1, IL-6, IL-1 β and NLRP3 mRNA were detected by real-time PCR. Three parallel replication wells were prepared for each sample, and the reaction was performed on the PCR instrument. TaqMan microRNA Analysis Kit (Applied Biosystems, Foster City, CA, USA) was used to measure gene expression level by 2 - ΔΔCt method. The primer series are shown in Table 1. GAPDH was used as an internal reference for detection.
- Tissue Sampling and Immunochemical staining
We obtained the gingival tissues from periodontitis patients (n=4, stage III/IV) and healthy control (n=3). All of the participants signed a consent form after confirming information about the study. All procedures are in accordance with the rules and requirements of the Human Research Ethics Committee of Ninth People's Hospital. All inclusion and exclusion criteria were listed in Table 2. The detailed procedures of gingival tissue collection and immunohistochemical staining were described in the previous study [16].
- Statistical methods and data analysis
The results in the study were expressed as mean ± standard deviation (M ± SD). First of all, we test the homogeneity of variance using Levene’s test by SPSS 25.0.0. The results showed that the variation within each population was equal (P > 0.05). So we used one-way ANOVA and Bonferroni post hoc test to evaluate the statistical significance between groups. All the experiments were repeated three times independently and then analyzed statistically (P < 0.05).