The Expression of miR-34a in Gingival Crevicular Fluid of Chronic Periodontitis and Its Connection with the TLR/NF-κB Signaling Pathway.

Objective: This study analyzed the expression of miR-34a in gingival crevicular fluid (GCF) of patients with chronic periodontitis and its connection with the Toll-like receptor (TLR)/nuclear factor kappa-B (NF-κB) signaling pathway.
Methods: We collected the GCF of the two groups of subjects, using RT-PCR to detect the expression of miR-34a and NF-κB p65 mRNA and TLR4 mRNA and ELISA to detect the inflammatory factor degree in GCF, and performed periodontal examinations on both groups.
Results: The gingival index, bleeding index, probe depth, and attachment loss indexes of periodontal examination in the observation group were remarkably superior to those in the control group (P < 0.05). The levels of IL-1β, IL-6, and TNF-α in the GCF of the observation group were higher than those of the control group (P < 0.05). The mRNA relative expression levels of miR-34a, NF-κB p65, and TLR4 in the GCF of the observation group were dramatically higher than those of the control group (P < 0.05). Correlation analysis showed that miR-34a was highly expressed in patients with chronic periodontitis.
Conclusion: There is an abnormally high expression of miR-34a in GCF of chronic periodontitis. Its expression is associated with the degree of periodontal inflammation, periodontal tissue damage, and the activation of the TLR/NF-κB signaling pathway and could be used as a potential index for auxiliary diagnosis and severity of the disease.

1. Introduction

Chronic periodontitis is a chronic oral illness induced by bacteria and pathogenic substances and is characterized by the invasion of gingiva and periodontal tissues. It will seriously affect the oral health of patients with the progress of the disease, and it is also an important cause of tooth loss in patients [1]. Chronic periodontitis is the most common form of periodontitis, usually causing swelling and redness in the gums. If it was not treated, it will lead to loss of soft tissue and bone. The gums will pull back from the teeth and eventually cause teeth to loosen and fall out.

In recent years, microRNA (miRNA) has been found to be abnormally expressed in varying inflammatory and neoplastic diseases, and miRNA detection is expected to become an important biomarker for the detection of inflammatory diseases [2]. Studies have found that a variety of miRNAs are involved in the regulation of periodontal tissue homeostasis and pathological processes and play a coordinating role in periodontal tissue inflammation and bone remodeling through different pathways [3, 4]. miR-34a is one of the members of the miR-34 family, and the coding gene is located in 1p36.23 of the chromosome. It can be induced by the tumor suppressor p53 and exert antitumor effects by inhibiting the expression of a variety of oncogenic factors downstream. miR-34a can also participate in various biological processes such as cell cycle, proliferation, senescence, apoptosis, and angiogenesis [5, 6]. Studies have verified [7] that miR-34a is involved in various inflammatory processes. The TLR/NF-κB signaling pathway participated in the regulative effect of a variety of inflammatory mediators and has been demonstrated in a variety of autoimmune diseases. To further realize the regulative function of miR-34a in disease, this study explored and analyzed the expression of miR-34a in GCF of chronic periodontitis and its correlation with the TLR/NF-κB signaling pathway. The report is as follows.

2. Materials and Methods

2.1. Research Objects

89 patients with chronic periodontitis admitted to our hospital from April 2018 to April 2020 were enrolled as the observation group, and 50 healthy volunteers that underwent physical checkups in our hospital during the same period were chosen as the control group. The study is approved by the institutional review board of the hospital. Written informed consent was obtained from each participant.

2.2. Inclusion and Exclusion Criteria

Inclusion criteria were as follows: (1) all patients in the observation group were diagnosed with moderate-to-severe chronic periodontitis, periodontal pockets ≥ 4 mm, attachment loss ≥ 3 mm, alveolar bone resorption ≥ 1/3, tooth loosening, probing bleeding, etc.; (2) the patient was diagnosed for the first time and has not received voluntary treatment before admission; (3) the age of patients is ≥35 years; and (4) the patient voluntarily signed the informed consent.

Exclusion criteria were as follows: (1) subjects who have combined oral local inflammatory diseases such as chronic pharyngitis or tonsillitis; (2) patients who have used antibacterial or immunosuppressive drugs, etc. within 3 months before admission; (3) women during pregnancy or lactation; (4) patients with malignant tumor or abnormal liver and kidney function; or (5) patients with an abnormal mental state that they cannot cooperate with this study.

2.3. Detection of miR-34a, NF-κB p65 mRNA, and TLR4 mRNA Expression in GCF

We removed the large plaque and large tartar in the GCF of patients and wiped the tooth surface and gingiva with a sterile cotton ball. We inserted the tip of no. 30 moisture-absorbent paper into the buccal periodontal pocket of the subject's teeth and retained it for 30 s (not used if contaminated by blood). We put the 4 quadrants of moisture-absorbing paper tips as one sample into the same EP tube, added with 200 μl PBS buffer and frozen in a refrigerator at -20°C for later use. The total RNA was extracted with TRIzol. We detected the concentration and purity of the total RNA with a spectrophotometer and reverse transcribed it into cDNA with a reverse transcriptase kit. The reaction conditions for RT-qPCR detection are as follows: 95°C 15 min predenaturation → 95°C 15 s and 60°C 60s → reaction, with a total of 40 cycles. The primers were synthesized by Shanghai Sangon Biotech Engineering Co., Ltd., with GAPDH as the internal reference gene. The primer sequences are shown in Table 1. The relative expression of each target gene is calculated by 2−△△Ct.

Table 1

Primer sequences.

Gene	PCR forward primer	Control primer R
miR-34a	5′-TGGCAGTGTCTTAGATGGTTGT-3′	5′-CTGTTCAGCTCGTACTGCATGTC-3′
NF-κB p65	5′-CCTGGATGACTCTTGGGAAA-3′	5′-CTTCGGTGTAGCCCATTTGT-3′
TLR4	5′-CAGAATGCTAAGGTTGCCGC-3′	5′-GTGGCTCGCTATCACCGTCT-3′
GAPDH	5′-AACAGCGACACCCACTCCTC-3′	5′-CATACCAGGAAATGAGCTTGACAA-3′

2.4. Detection of Inflammatory Cytokines

After taking samples of GCF from the two groups of subjects according to the above method, an enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Invitrogen) was used to measure the inflammatory cytokine degree, including interleukin-1β (IL-1β) and interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The details of the inflammatory cytokines are shown in Results.

2.5. Periodontal Examination

2.5.1. Gingival Index (GI)

The inflammation degree was recorded in four areas: lingual marginal gingiva, mesial buccal gingival papilla, buccal margin gingiva, and buccal distal gingival papilla. The inflammation was rated on a four scale: 0 point (normal gingiva), 1 point (mild inflammation), 2 points (moderate inflammation), and 3 points (severe inflammation). The score of each tooth and the examinee (the sum of the scores of the 4 teeth examined) was calculated.

2.5.2. Bleeding Index (BI)

The bleeding situation was rated on a six-point scale (0 point (healthy gingiva), 1 point (inflammatory changes in gingival color, but no bleeding on probing), 2 points (spotty bleeding after probing), 3 points (bleeding occurred after probing and spread along the gingival margin), 4 points (the bleeding filled and overflowed the gingival sulcus), 5 points (the subject with spontaneous bleeding)).

2.5.3. Probing Depth (PD)

Each test tooth was recorded in 6 locations (lingual mesial, centre, distal and buccal mesial, centre, and distal). The probing depth of the sulcus of the healthy gingiva did not exceed 2-3 mm.

2.5.4. Attachment Loss (AL)

When the gingiva was not receding, the loss of attachment was the difference between the probing depth of the periodontium and the distance from the cementoenamel junction (CEJ) to the gingival margin, and when the gingiva retracted, the attachment loss was the sum of probing depth and the distance from CEJ to the gingival margin.

2.6. Statistical Method

The statistical analysis of the research was conducted by SPSS 25.0. The comparison of measurement data was carried out by the t-test, and the comparison of enumeration data was carried out by the χ2 test, and the correlation was analyzed by Pearson correlation. P < 0.05 is referred to as the statistically significant difference.

3. Results

3.1. Clinical Data of the Patients

There are a total of 89 cases in the observation group and 50 in the control group. The average age in the observation group was 48.59 ± 7.83 yd, while it was 47.39 ± 8.42 yd in the control group. The systolic blood pressure in the observation group was 120.85 ± 11.87 mmHg, and it was 119.74 ± 13.42 mmHg in the control group. Diastolic blood pressure was 75.62 ± 7.39 mmHg in the observation group and 73.96 ± 7.10 mmHg in the control group. BMI was 23.17 ± 3.31 kg/m2 in the observation group and 23.46 ± 3.04 kg/m2 in the control group. There was no statistically significant difference in clinical data between the gender, age, systolic blood pressure, diastolic blood pressure, and BMI (P > 0.05) (Table 2).

Table 2

Comparison of clinical data between the two groups.

Group	Number of cases	Gender		Age (yd, x¯±s)	Systolic blood pressure (mmHg, x¯±s)	Diastolic blood pressure (mmHg, x¯±s)	BMI (kg/m2, x¯±s)
		Male	Female
Observation group	89	51	38	48.59 ± 7.83	120.85 ± 11.87	75.62 ± 7.39	23.17 ± 3.31
Control group	50	29	21	47.39 ± 8.42	119.74 ± 13.42	73.96 ± 7.10	23.46 ± 3.04
t/χ2	—	0.006		0.844	0.505	1.289	0.501
P	—	0.936		0.400	0.615	0.200	0.611

3.2. Periodontal Examination Results of the Two Groups of Subjects

The indexes of the gingiva, bleeding, penetration depth, and attachment loss of periodontal examination in the observation group were remarkably superior to those in the control group (P < 0.05) (Table 3).

Table 3

Comparison of periodontal examination results between the two groups of subjects ().

Group	Number of cases	GI	BI	PD (mm)	AL (mm)
Observation group	89	1.91 ± 0.26	3.97 ± 0.93	5.12 ± 1.64	5.89 ± 1.73
Control group	50	0.64 ± 0.19	1.74 ± 0.46	2.08 ± 0.53	0.93 ± 0.36
t	—	30.276	15.881	12.722	20.001
P	—	<0.001	<0.001	<0.001	<0.001

3.3. Comparison of Inflammatory Levels in GCF between the Two Groups

The levels of IL-1β, IL-6, and TNF-α in the GCF of the observation group were higher than those of the control group (P < 0.05) (Table 4).

Table 4

Comparison of inflammatory level in GCF between the two groups ().

Group	Number of cases	IL-1β (pg/ml)	IL-6 (pg/ml)	TNF-α (ng/ml)
Observation group	89	3.74 ± 0.67	5.78 ± 0.77	2.15 ± 0.35
Control group	50	2.16 ± 0.33	3.49 ± 0.54	0.98 ± 0.16
t	—	15.627	18.603	22.336
P	—	<0.001	<0.001	<0.001

3.4. Comparison of miR-34a, NF-κB p65 mRNA, and TLR4 mRNA Expression in GCF between the Two Groups

The relative expression of miR-34a, NF-κB p65, and TLR4 mRNA in the GCF of the observation group was dramatically higher than that of the control group (P < 0.05) (Table 5).

Table 5

Comparison of miR-34a, NF-κB p65, and TLR4 mRNA expression in GCF between the two groups ().

Group	Number of cases	miR-34a	NF-κB p65 mRNA	TLR4 mRNA
Observation group	89	1.472 ± 0.331	1.682 ± 0.402	1.552 ± 0.397
Control group	50	0.978 ± 0.216	1.023 ± 0.295	1.072 ± 0.319
t	—	9.473	10.151	7.321
P	—	<0.001	<0.001	<0.001

3.5. Correlation Analysis

The results of correlation analysis showed that in chronic periodontitis, the miR-34a expression level was notably positively correlated with GI, BI, PD, AL, IL-1β, IL-6, TNF-α, NF-κB p65 mRNA, and TLR4 mRNA in GCF of chronic periodontitis (P < 0.05) (Table 6 and Figure 1).

Table 6

Correlation analysis.

Index	miR-34a
	r	P
GI	0.397	<0.001
BI	0.406	<0.001
PD	0.385	<0.01
AL	0.448	<0.01
IL-1β	0.559	<0.001
IL-6	0.529	<0.001
TNF-α	0.518	<0.001
NF-κB p65 mRNA	0.492	<0.001
TLR4 mRNA	0.447	<0.001

Figure 1

Correlation analysis. The correlation of miR-34a expression level with GI, BI, PD, AL, IL-1β, IL-6, TNF-α, NF-κB p65 mRNA, and TLR4 mRNA in GCF of chronic periodontitis was analyzed.

4. Discussion

Chronic periodontitis is the main cause of loosening and loss of teeth in adults. A variety of complex factors can affect the occurrence, progression, and prognosis of periodontal disease [8-10]. GCF is an inflammatory exudate that penetrates from gingival connective tissue through the epithelium in the sulcus into the gingival sulcus. GCF can reflect the metabolic changes of periodontal supporting tissue, and its level is a sensitive indicator for evaluating the degree of periodontal inflammation [11, 12].

The current focus and hotspot of research on chronic periodontitis are to find factors that are closely related to the diagnosis and evaluation of the disease. miRNA is a highly conserved noncoding RNA, which is paired with the target gene mRNA to degrade or inhibit its translation to achieve negative regulation of gene expression. miRNA is a highly conserved noncoding RNA, which can degrade or inhibit its translation after complementary pairing with target gene mRNA to achieve negative regulation of gene expression. The detection of miRNA has become an important biological marker for the diagnosis and monitoring of varying diseases [13-15]. miR-34a is a pivotal regulative factor of natural and innate immunity. Studies have found that miR-34a expression is increased in chronic rheumatoid arthritis, which weakens the inhibitory effect of Th1 cell differentiation, thus sustaining the chronic inflammatory response [16-18]. The functions of miR-34a in the occurrence and progression of chronic periodontitis remain unclear at the moment. This essay discussed and analyzed the expression of miR-34a in GCF of patients with chronic periodontitis, as well as its connection with the TLR/NF-κB signaling pathway. The study findings revealed that miR-34a relative expression in GCF of the observation group was critically higher than that of the control group, suggesting an abnormal expression of miR-34a in chronic periodontitis.

Chronic periodontitis is an inflammatory and destructive illness induced by plaque microorganisms. The inflammatory cells enter the periodontal tissue under the action of chemokines and mediate the production of various inflammatory mediators [19-21]. TNF-α can promote the production of chemokines, adhesion molecules, inflammatory factors, etc. and participate in the initiation of chronic periodontitis [22]. This research results showed that the levels of IL-1β, IL-6, and TNF-α in the GCF of the observation group were remarkably higher than those of the control group, which is consistent with the conclusion by scholars [23, 24] and suggests the existence of gingival crevicular inflammation in chronic periodontitis. The expression level of miR-34a was positively correlated with the levels of IL-1β, IL-6, and TNF-α in GCF and the periodontal clinical symptom indicators (GI, PD, AL, and PLI), confirming that miR-34a could objectively reflect the degree of the gingival crevicular inflamed condition and the severity of disease in chronic periodontitis.

The TLR/NF-κB signaling pathway exerts a crucial action in the process of inflammatory and immune response in the body and is connected with the immunopathological mechanism of a variety of diseases such as rheumatoid arthritis [25, 26]. In this study, we used RT-PCR to detect the expression of NF-κB p65 mRNA and TLR4 mRNA in GCF of chronic periodontitis and subjects in the normal state, respectively. The results showed that the relative expression levels of NF-κB p65 mRNA and TLR4 mRNA in GCF of the observation group were higher than those of the control group and were positively correlated with the expression of miR-34a. This is consistent with the results of scholars [27, 28], confirming that there is an activation of the TLR/NF-κB signaling pathway in GCF of chronic periodontitis and further demonstrating that miR-34a can objectively reflect the degree of periodontal tissue damage in chronic periodontitis.

The disadvantage of this study was to demonstrate that high expression of miR-34a is associated with the degree of periodontal inflammation and periodontal tissue damage and could be used as a potential index for auxiliary diagnosis and severity of the disease. However, there are also limits. First, the number of patients is not so large. Second, the mechanism was not clarified, which needs further studies to demonstrate this.

5. Conclusion

In summary, there is an abnormally high expression of miR-34a in GCF of chronic periodontitis. Its expression is correlated with the degree of periodontal inflammation, periodontal tissue damage, and the activation of the TLR/NF-κB signaling pathway and could be used as a potential index for auxiliary diagnosis and severity of the disease.