Correlation analysis of cell-free DNA in gingival crevicular fluid with periodontal clinical indicators and cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway

doi:10.7518/hxkq.2025.2024391

Abstract

Abstract:

Objective This study aims to explore the potential relationships of cell-free DNA (cfDNA) in gingival crevicular fluid (GCF) with periodontal clinical indicators and the expression of DNA receptor pathway cyclic guanosine phosphate-adenosine phosphate synthase (cGAS)-stimulator of interferon genes (STING) in gingival tissues and human gingival fibroblasts (HGFs). Methods GCF and gingival tissue samples were collected from periodontally healthy individuals and patients diagnosed with periodontitis. Periodontal clinical indicators were recorded, including plaque index (PLT), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). The concentration of cfDNA in GCF was quantified, and the correlation between GCF and periodontal clinical indicators was analyzed. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the distribution of cGAS, STING, and p-STING in gingival tissues. Additionally, the mRNA expression levels of the key components of the cGAS-STING signaling pathway, namely, cGAS, STING, inhibitory of kappa-B kinase (IKK), nuclear factor kappa-B p65 (NF-κB p65), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured. Furthermore, cfDNA extracted from GCF was employed to stimulate HGFs in the healthy control and periodontitis groups, and the mRNA expression levels of the key molecules of cGAS-STING signaling pathway were detected through Western blot and RT-qPCR. Results The concentration of cfDNA in GCF was found to be significantly elevated in the periodontitis group compared with the control group. Moreover, cfDNA concentration demonstrated a strong positive correlation with the periodontal clinical indicators. Immunofluorescence analysis revealed considerably increased percentage of fluorescence co-localization of cGAS, STING, and p-STING with the gingival fibroblast FSP-1 marker in the gingival tissues of the periodontitis group. The mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6,and TNF-α were significantly higher in the periodontitis group. In vitro stimulation of HGFs with GCF-derived cfDNA resulted in increased protein expression of cGAS and p-STING and considerably upregulated the mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6, and TNF-α in the healthy and periodontitis groups compared with the blank group. Correlation analysis showed that the concentration of cfDNA at the sampling site was positively correlated with the mRNA expression levels of cGAS, STING, NF-κB p65, and IL-6 in gingival tissues. Conclusion cfDNA concentrations in the GCF of patients with periodontitis are considerably elevated, and are associated with the activation of the cGAS-STING signaling pathway in HGFs. These findings suggest that cfDNA contributes to the progression of periodontitis.

Key words: periodontitis, cell-free DNA, cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway, human gingival fibroblasts

CLC Number:

R781.4

Chen Lan, Zhu Xuanzhi, Zhou Jieyu, Li Jiyao, Zhao Lei. Correlation analysis of cell-free DNA in gingival crevicular fluid with periodontal clinical indicators and cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway[J]. West China Journal of Stomatology, 2025, 43(6): 808-818.

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基因	引物序列（5’-3’）	产物大小/bp
cGAS	F：GCTGCTCCACGAAGCCAAGAC	194
	R：GTCCACAACCCCTTTCACCATCC
STING	F：AGGAGAGCCACCAGAGCACAC	231
	R：ACCGCATTTGGGAGGGAGTAGTAG
IKK	F：GCGGTCCGACGTATGTGGTAAC	119
	R：TGTGCTGCTGCTGCGATGC
NF-κB p65	F：CGCATCCAGACCAACAACAACC	80
	R：TGGAAGCAGAGCCGCACAG
IL-6	F：GCCTTCGGTCCAGTTGCCTTC	133
	R：GTTCTGAAGAGGTGAGTGGCTGTC
TNF-α	F：TGGCGTGGAGCTGAGAGATAACC	149
	R：TCTGGTAGGAGACGGCGATGC
IL-1β	F：GGACAGGATATGGAGCAACAAGTGG	123
	R：CAACACGCAGGACAGGTACAGATTC
GAPDH	F：CACCCACTCCTCCACCTTTGAC	112
	R：GTCCACCACCCTGTTGCTGTAG

临床指标		健康组（n=10）	牙周炎组（n=10）
性别	男	5	4
	女	5	6
年龄/岁		24.00±1.48	30.20±3.87^***
BMI/（kg/m²）		21.06±1.72	21.53±2.19
PLI	全口	0.16±0.08	0.74±0.29^****
	取样位点	0.18±0.25	0.93±0.43^***
BI	全口	0.06±0.03	2.32±0.61^****
	取样位点	0.13±0.17	3.55±0.48^****
PD/mm	全口	1.90±0.19	4.40±0.85^****
	取样位点	2.35±0.25	9.35±0.98^****
CAL/mm	全口	0	4.31±1.57^****
	取样位点	0	8.95±1.81^****
GCF cfDNA/（mg/mL）		1.76±1.09	13.45±2.06^****

项目	年龄	BMI	PLI		BI		PD		CAL
项目	年龄	BMI	全口	取样位点	全口	取样位点	全口	取样位点	全口	取样位点
F值	15.750	0.258 0	16.140	12.440	80.000	97.990	88.810	133.100	115.100	173.000
r值	0.467	0.014 2	0.473	0.409	0.817	0.845	0.832	0.881	0.865	0.906
P值	0.000 9	0.617 4	0.000 8	0.002 4	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1

组别	cGAS	STING	IKK	NF-κB p65	IL-1β	IL-6	TNF-α
健康组	1.06±0.40	0.77±0.24	1.69±1.48	0.88±0.15	1.21±0.71	1.15±0.77	0.79±0.33
牙周炎组	2.63±0.81^**	2.17±0.57^**	11.49±8.30^*	1.84±0.47^**	2.47±0.77^*	16.75±8.82^**	2.36±1.21^**