Secretory microRNA-29 expression in gingival crevicular fluid during orthodontic tooth movement

Abstract

Secretory microRNAs (miRNAs) have been used increasingly as biomarkers for cancers, autoimmune diseases and inflammatory diseases. They are reported as being freely circulated or encapsulated in microvesicles such as exosomes. This study was performed to elucidate the presence of miRNAs with exosomes in human gingival crevicular fluid (GCF), and the expression profile of miRNA-29 during orthodontic tooth movement. Four healthy volunteer and fifteen orthodontic patients were enrolled in the study. Secretory miRNA in GCF was collected and analyzed using a bioanalyzer, realtime PCR and Western blot analysis. The expression profile of secretory miR-29 family in GCF was analyzed during the course of canine retraction for 6 weeks. The results demonstrated the presence of miRNAs in the GCF. After series of ultracentrifugation and RT-PCR array, exosome-depleted fractions and pellets were isolated and we found that secretory miRNAs were detected in both the exosome-associated fraction and the exosome-depleted supernatant fraction; however, the concentration of miRNAs was higher in the exosome-associated fraction than in the exosome-depleted fraction suggesting a close association between the secretory miRNAs and exosomes in GCF. We also demonstrated the increased expression profiles of miR-29 family during six weeks of orthodontic tooth movement in humans. Secretory miRNAs are present in GCF and secretory miRNA-29 family expression profiles increase during the tooth movement in humans. Secretory miRNA-29 in GCF could serve as potential biomarkers for periodontal remodeling.

Fig 1. Diagram of human crevicular fluid collecting time.

T0: prior to bonding the fixed orthodontic appliances, T1: on the day of canine retraction, 60 minutes after engaging the elastomeric powerchains onto the canine bracket. T2: 24 hours after initiation of canine retraction, T3: 7 days after initiation of canine retraction, T4: 6 weeks after initiation of canine retraction.

Fig 2. Presence of miRNA in healthy human crevicular fluid.

“gel-like” images of R6K ScreenTape (A) after subjected to Tapestation bioanalyzer showed RNA ladder (L) on left lane and only small-sized RNA present in the sample lanes (middle and right lane). The positive bands approximately 50 nucleotides (nt) were present in representative GCF samples (A1 and B1). Electropherogram (B) corresponding to the gel-like images on the (A) figure. The x-axis on the electropherogram represents RNA size (nt), while the y-axis represents the measurement response of fluorescence units (FUs).

Fig 3. Electron microscope images and Western blot of isolated exosome from healthy human gingival crevicular fluid.

Electron microscopy (A and B) of the ultracentrifugation pellet from GCF shows the characteristic spherical shape and size (50-100nm) of exosomes, Western blot (C) shows strong staining of the ultracentrifugation pellet with the exosomal membrane markers anti-CD63 and anti-CD9.

Fig 4. Distribution of miRNAs in exosome pellet and exosome-depleted supernatant fractions from crevicular crevicular fluid.

Crevicular fluid miRNAs in all subjects are predominantly in exosomes; however, some miRNAs are present in exosome-depleted supernatant (A). The higher CT cycles are detected in exosome-depleted supernatant than in exosome pellet after normalized with amount of RNA (ng) (B) and the ΔCT is the difference between CT of supernatant-CT of exosome pellet after normalization with let-7d and g. Positive numbers show higher concentrations in the exosome pellet whereas negative numbers indicate higher concentrations in the exosome-depleted supernatant (C).

Fig 5. The expression profile of miRNA-29 family in GCF during tooth movement in human.

The expression of miRNA-29a, -29b, and -29c were shown as gradually increase profile toward the last timepoint (6-wk). The significant differences are detected between T0 (pretreatment) and T4 (6-wk) in all studied miRNAs. Note that the significant differences of miRNA-29b expression are detected between T0 and T1 (1-hr), T0 and T3 (7-day) (P<0.05).