Role of SIRT6 in Chronic Periodontitis and Osteogenic Differentiation of Periodontal Ligament Stem Cells

Abstract

Objective: Chronic periodontitis (CP) is a common inflammatory disease that may cause systemic conditions. This study aimed to explore the correlation between serum Sirtuin 6 (SIRT6) levels and clinical characteristics of CP and the mechanism by which SIRT6 regulates osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs).

Methods: The serum expression of SIRT6 and enhancer of zeste homolog 2 (EZH2) in CP patients and healthy controls was measured by real-time quantitative polymerase chain reaction. The correlation between SIRT6 expression, clinical characteristics of CP [probing pocket depth/attachment level/bleeding on probing (PD/AL/BOP)], and EZH2 expression was assessed by Pearson analysis. hPDLSCs were cultured in adipogenic and osteogenic induction media, followed by measurement of adipogenic and osteogenic differentiation abilities. The levels of proinflammatory factors and cell viability in lipopolysaccharide-treated hPDLSCs were measured. After osteogenic induction of hPDLSCs, SIRT6 and EZH2 levels were detected. The enrichment of SIRT6 and H3K9ac on the EZH2 promoter was examined.

Results: SIRT6 was decreased in CP patients. SIRT6 expression was negatively correlated with PD/AL/BOP in CP patients and EZH2 expression. In the inflammatory microenvironment, osteogenic differentiation of hPDLSCs was suppressed, and SIRT6 expression was decreased. SIRT6 overexpression promoted osteogenic differentiation of hPDLSCs and alleviated inflammation. SIRT6 suppressed EZH2 expression by deacetylating H3K9ac. Overexpression of EZH2 partially reversed the promotive effect of SIRT6 overexpression on osteogenic differentiation of hPDLSCs under the inflammatory microenvironment.

Conclusions: SIRT6 is negatively correlated with clinical characteristics of CP. SIRT6 is downregulated in hPDLSCs under the inflammatory microenvironment, and overexpression of SIRT6 promotes osteogenic differentiation of hPDLSCs by suppressing EZH2 expression through deacetylation of H3K9ac.

Keywords: Chronic periodontitis; EZH2; Human periodontal ligament stem cells; Osteogenic differentiation; SIRT6; Serum.

Fig. 1

SIRT6 is poorly expressed in the serum of CP patients and has high diagnostic value for CP. A, qRT-PCR was used to detect the expression of SIRT6 in the serum of HC group (N = 70) and CP group (N = 70); B, ROC curve analysis of the diagnostic performance of SIRT6 for CP patients. Data in panel A were analysed by independent samples t test, and data in panel B were analysed by ROC curve analysis. ** P < .01. CP, chronic periodontitis; HC, healthy control; qRT-PCR, real-time quantitative polymerase chain reaction; ROC, receiver operating characteristic; SIRT6, Sirtuin 6.

Fig. 2

The serum expression of SIRT6 is negatively correlated with PD, AL, BOP, and PI. A, The correlation between serum SIRT6 expression and PD. B, The correlation between serum SIRT6 expression and AL. C, The correlation between serum SIRT6 expression and BOP. D, The correlation between serum SIRT6 expression and PI; N = 70. Data in panels A-D were analysed by Pearson analysis. AL, attachment level; BOP, bleeding on probing; PD, probing pocket depth; PI, plaque index; SIRT6, Sirtuin 6.

Fig. 3

Morphological observation and identification of hPDLSCs. A, Morphological observation of hPDLSCs under the microscope. B, Oil Red O staining to detect adipogenic differentiation of hPDLSCs. C, Alizarin Red S staining to detect osteogenic differentiation of hPDLSCs. D, Flow cytometry to detect the levels of surface markers of hPDLSCs (positive: CD29, CD90; negative: CD34, CD45). hPDLSCs, human periodontal ligament stem cells.

Fig. 4

Overexpression of SIRT6 promotes osteogenic differentiation of hPDLSCs in an inflammatory microenvironment. hPDLSCs were transfected with overexpressing SIRT6 plasmid (oe-SIRT6), and empty plasmid (oe-NC) was used as a control. Transfection efficiency was detected 48 hours posttransfection, followed by treatment with 10 μg/mL LPS for 24 hours to simulate the inflammatory microenvironment. A, ELISA was used to detect the levels of TNF-α, IL-6, and IL-1β in the cells. B, CCK-8 assay was employed to measure cell viability. C, ALP staining and ALP activity assay. D, Alizarin Red S staining and mineralised nodule detection. E, qRT-PCR was used to detect the transfection efficiency of oe-SIRT6 plasmid. F-G, qRT-PCR or Western blot assay was used to detect the expression of SIRT6 in the cells. Independent experiments were repeated 3 times, and the data are presented as mean ± SD. Data in panel E were analysed by t test. Data in panel A were analysed by two-way ANOVA, and data in panels B/C/D/F/G were analysed by one-way ANOVA, followed by Tukey's multiple comparisons test; * P < .05, ** P < .01. ALP, alkaline phosphatase; ANOVA, analysis of variance; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; hPDLSCs, human periodontal ligament stem cells; IL, interleukin; LPS, lipopolysaccharide; qRT-PCR, real-time quantitative polymerase chain reaction; SIRT6, Sirtuin 6; TNF-α, tumour necrosis factor-α.

Fig. 5

SIRT6 inhibits EZH2 expression through H3K9 deacetylation. A, qRT-PCR was performed to detect the expression of EZH2 in the serum of HC group (N = 70) and CP group (N = 70). B, Pearson analysis of the correlation between the expression of SIRT6 and EZH2 in the serum of CP patients (N = 70). C-D, qRT-PCR or Western blot was used to detect the levels of EZH2 in hPDLSCs. E, ChIP was used to detect the enrichment of SIRT6 and H3K9ac in the EZH2 promoter region. Independent experiments were repeated 3 times, and the data are presented as mean ± SD. Data in panel A were analysed by t test. Data in panel B were analysed by Pearson analysis. Data in panels C/D were analysed by one-way ANOVA, and data in panel E were analysed by two-way ANOVA, followed by Tukey's multiple comparisons test; ** P < .01. ANOVA, analysis of variance; CP, chronic periodontitis; ChIP, chromatin immunoprecipitation; EZH2, enhancer of zeste homolog 2; H3K9ac, lysine 9 acetylation; HC, healthy control; hPDLSCs, human periodontal ligament stem cells; qRT-PCR, real-time quantitative polymerase chain reaction; SIRT6, Sirtuin 6.

Fig. 6

Overexpression of EZH2 partially reverses the promotive effect of SIRT6 overexpression on osteogenic differentiation of hPDLSCs in an inflammatory microenvironment. hPDLSCs were transfected with overexpressing EZH2 plasmid (oe-EZH2), and empty plasmid (oe-NC) was used as a control. Transfection efficiency was detected 48 hours posttransfection, followed by treatment with 10 μg/mL LPS for 24 hours to simulate the inflammatory microenvironment. A, qRT-PCR was used to detect the transfection efficiency of oe-EZH2 plasmid. B-C, qRT-PCR or Western blot was used to detect the expression of EZH2 in the cells. D, ELISA was employed to detect the levels of TNF-α, IL-6, and IL-1β in the cells. E, CCK-8 assay was used to measure cell viability. F, ALP staining and ALP activity assay. G, Alizarin Red S staining and mineralised nodule detection. Independent experiments were repeated 3 times, and the data are presented as mean ± SD. Data in panel A were analysed by t test. Data in panels B/C/E/F/G were analysed by one-way ANOVA, and data in panel D were analysed by two-way ANOVA, followed by Tukey's multiple comparisons test; * P < .05, ** P < .01. ALP, alkaline phosphatase; ANOVA, analysis of variance; CCK-8, cell counting kit-8; ELISA, enzyme-linked immunosorbent assay; EZH2, enhancer of zeste homolog 2; hPDLSCs, human periodontal ligament stem cells; IL, interleukin; LPS, lipopolysaccharide; qRT-PCR, real-time quantitative polymerase chain reaction; SIRT6, Sirtuin 6; TNF-α, tumour necrosis factor-α.

Fig. 7

Overexpression of SIRT6 inhibits EZH2 expression by removing the acetylation modification on histone H3K9, thereby promoting osteogenic differentiation of hPDLSCs in an inflammatory microenvironment. EZH2, enhancer of zeste homolog 2; hPDLSCs, human periodontal ligament stem cells; SIRT6, Sirtuin 6.