Expression And Clinical Significance Analysis Of MiRNA-146a In Saliva Of Patients With Chronic Periodontitis

The World Health Organization had alread listed oral health as one of the ten criteria for human health, and moreover periodontal disease is one of the two major oral diseases.Chronic periodontitis is the most common type of periodontal disease, accounting for more than 95%, while its clinical features are periodontal pockets formation, attachment loss, alveolar bone loss, teeth loose, and finnally may eventually leads to tooth loss, which is the leading cause of tooth loss in adults. Chronic periodontitis is a multifactorial disease which process involes a complex interaction of microbes and host factors, so we can know host response to bacterial invasion determines the occurrence, severity, range size, development speed and others, and currently its exact pathogenesis hasnot yet entirely clear.Saliva contains a large number of molecules presenting in the blood, such as proteins, RNA, DNA, and other viral particles. So saliva has been hailed as " a mirror of human health ", it can suggest a variety of disease states of the body, such as infectious diseases, cancer and cardiovascular diseases. In recent years, researches on saliva testing and disease diagnosis increased, so saliva miRNA as a novel marker gradually is attented by researchers. In recent years research had also reported that tissue, plasma and saliva had similar miRNA expression profiling, that is to say a plurality of kinds of miRNA expression occurred significant change in tissue, blood and saliva. At present, miRNAs expression of salivary in chronic periodontitis has not been studied at home and abroad.miRNA is endogenous, highly conserved sequences, about 22 nucleotides, single-stranded and non-coding small RNA. miRNA complementary pairing with gene mRNA 3’untranslated region (3’UTR) which resulted in mRNA degradation or translational repression, and thus regulation of gene expression were negative in the post-transcriptional level. With the continuous development of RNA genomics and bioinformatics prediction, there are more than 530 species of miRNA in the human body were discovered, and had an important role in human disease development. Researchers detected that many kinds of miRNA molecules are detected stable existence in the saliva of patients with oral squamous cell carcinoma, and fluorescence quantitative PCR results showed that miRNA-200a and miRNA-125a expression in the saliva of patients with oral squamous cell carcinoma was significantly reduced. Wu weidong etc. detected miRNA expression profiles in the saliva of esophageal cancer patients and healthy controls, and found that miRNA-144 in the saliva of patients with esophageal cancer highly expressed, which can be used as an early diagnosis of esophageal cancer gene markers. Xie et al found that miRNA-3679-5p and miRNA-940 in the saliva can be acted as a potential non-invasive pancreatic cancer diagnosis markers.miRNA has clearly played a regulatory role in the immune system, which miRNA-146a is the first found closely related immune inflammation. It is a typical multi-function gene, involved in the occurrence and development of a variety of physiological and pathological processes under mediation of downstream genes, such as immune, inflammation, hematopoiesis and cancer. Human miRNA-146 is composed by 22 nucleotides, including miRNA-146a and miRNA-146b in two forms, miRNA-146a gene located on LOC285628 human chromosome 5, the mature sequence in the second exon. miRNA-146a is highly expressed in monocytes, T lymphocytes and macrophages, and other immune and inflammatory cells. Recently, Xie and others detected by microarray analysis that some miRNA including miRNA-146a upregulated in the gingival. In addition, the study also found that miRNA-146a and its target gene polymorphisms were related to the susceptibility of chronic periodontitis.From the above, we hypothesized that miRNA-146a may be involved in the pathogenesis of chronic periodontitis, but the mechanism is unclear. This study will detect the miRNA-146a expression levels in saliva supernatant of chronic periodontitis and healthy controls by stem-loop RT-PCR, and analyze the relationship between expression levels and periodontosis degrees; campare miRNA-146a expression levels before and after the basic periodontal treatment, and to explore its relationship between periodontal clinical parameters and the secretion of inflammatory cytokines, TNF-α, IL-1β, IL-6, IL-8 and IL-10, then providing the preliminary information about the assessment of basic periodontal therapy and the prediction of disease progression. This paper is divided into two parts contentPart I Detection of miRNA-146a and related factors expression in the salivary of chronic periodontitis1. ObjectThis study prepares to detect miRNA-146a expression levels in the salivary supernatant of chronic periodontitis patients and healthy controls by stem-loop real-time quantitative PCR, then anlyze the relationship among its expression levels, periodontal clinical indicators and secretion of inflammatory cytokinesTNF-a, IL-1β, IL-6, IL-8, IL-10, in order to provide a reference for clinical reatment of chronic periodontitis.2. Methods(1) SubjectsAccording to diagnostic criteria of chronic periodontitis of the 1999 US Periodontology classification workshop, sixty four chronic periodontitis patients (periodontosis group) who were admitted into Department of Stomatology, Southwest Hospital from January 2012 to may 2014 were investigated, including 38 males and 26 females, with an average age of 44 years old. While Choose 30 healthy volunteers as the control group, including 17 males,13 women, average age 42 years.(2) periodontal examination and groupPeriodontal clinical parameters were recorded:gingival index (gingival index, GI), probing depth (probing depth, PD), attachment loss (attachment loss, AL) and plaque index (plaque index, PLI). According to probing depth, gingival index, attachment loss and the extent of alveolar bone absorption showed by X-ray to measure the degree of periodontitis, grouped as follows① healthy control group:gingival index=0, no attachment absorption of alveolar bone loss and periodontal probing depth<3mm, a total of 30 cases.② mild periodontitis:gingival index> 1, attachment loss 1～2 mm, alveolar bone length<1/3 of root length, periodontal probing depth≤4 mm, tooth mobility is not obvious, a total of 18 cases.③ moderate periodontitis:gingival index> 1, attachment loss 3～4mm, alveolar bone absorption length of root length 1/3 to 1/2 long, probing depth≤6mm, multi-rooted teeth with light furcation, slightly loose tooth, a total of 24 cases.④ severe periodontitis:gingival index> 1, attachment loss≥5mm, alveolar bone length> 1/2 root length, periodontal probing depth> 6mm, furcation obviously, loose tooth, a total of 22 cases.(3) saliva collectionSaliva samples collected early in the morning by cotton bud,2h before collecting subjects should had no eating, drinking, smoking, strenuous exercising and mood fluctuation. The subjects take seat, head slightly forward position, using cotton bud with citric acid to wipe the mouth in order to increase the secretion of saliva, after 1.5min collecting the saliva by 10ml tube with RNA enzyme-free sterile centrifuge tube. Immediately take saliva supernatant after 4℃,3000r/min centrifugal 15min. The collected supernatant was frozen at-80℃ for reserving.(4) Detection of miRNA-146a expression in saliva supernatant1ml saliva supernatant were used to extracting and purifying total RNA with mirVana PARIS Kit, and extraction step reference kit instructions. Using stem-loop primer to have reverse transcription with an internal reference of miRNA-16. miRNA-146a in total RNA reverse transcribed into cDNA with TaKaRa Company PrimeScript RT reagent Kit reverse transcription kit. Reverse transcription product frozen at-80℃ for reserving. RT-qPCR detection was carried out according to SYBR Premix Ex TaqTM PCR reaction kit with ABI StepOne Plus real-time quantitative PCR (Real-time PCR, the RT-qPCR instrument. Total volume of reaction was 20μl, and each specimen made three wells. Calculate the sample average cycle threshold (cycle threshold, Ct), the miRNA-146a Ct value minus miRNA-16 Ct value as ΔCt value. RT-qPCR results were analyzed by relative quantification method, calculate2-ΔCt normalized value to represents the relative levels of miRNA-146a expression.(5) Detection of TNF-a, IL-1β,IL-6, IL-8 and IL-10 Levels in saliva supernatant Using ELISA reaction assay (ELISA) to detect TNF-a, IL-1β, IL-6, IL-8 and IL-10 levels in the saliva supernatant samples, according to instructions:the standards were diluted to the ELISA reaction plate, each ELISA plate setup 8 standard holes; draw 100μl sample to ELISA reaction plate, mix gently,37℃ incubated for 2h; draw first antibody solution to 100μl ELISA reaction holes, mix lightly,37℃ incubated 1h; Microplate Washer washed 5 times, each 45s; after washing added 100μl of horseradish peroxidase-conjugated secondary antibody in ELISA wells, mix gently,37℃ incubated for lh; Microplate Washer washed five times, each 45s; after washing washer fluid substrates A and B were added to each well 50μl respectively, mix gently,37℃ incubated for 30min; adding 50μl termination solution, mix gently; OD values of each well were measured at 450nm wavelength; standard concentration as abscissa, corresponding OD value as ordinate to carry out curve fitting, according to the standard curve fitting calculated the purity of cytokines each test sample.(6) Statistical analysisUsing SPSS 17.0 statistical software to analyze results. Measurement data were showed by x±s, and the groups were compared using LSD-t test, with P<0.05 considering statistically significant. The relationship among miRNA-146a expressions, periodontal clinical indicators and cytokine levels were analyzed by the Pearson correlation coefficient.3. Results(1) miRNA-146a expression (2.72±0.81) in chronic periodontitis saliva supernatant was significantly higher than its in controls (0.82±0.36), and the difference was statistically significant (P<0.01).(2) miRNA-146a expression levels of mild chronic periodontitis group was 1.92±0.44, moderate periodontitis group was 2.71±0.58, severe periodontitis group was 3.40±0.62. It is obvious that miRNA-146a expression in the light periodontitis, moderate periodontitis, and severe periodontitis was significantly higher (P<0.01).(3) miRNA-146a expression levels and gingival index, probing depth, attachment loss and plaque index in chronic periodontitis showed a significant positive correlation, r=0.752, P<0.001; r=0.657, P<0.001; r=0.586, P<0.001; r=0.647, P<0.001.(4) in patients with chronic periodontitis the averages of TNF-a, IL-1β, IL-6, IL-8 were 9.46±6.72 pg/ml,48.20±14.97 pg/ml,6.26±4.05 pg/ml,145.97±43.68 pg/ml, all significantly higher, and the difference was statistically significant (P<0.01). The IL-10 levels (mean of 6.74±4.31 pg/ml) was significantly lower than the control group, and the difference was statistically significant (P<0.05).(5) In chronic periodontitis saliva supernatant miRNA-146a expression levels and TNF-a, IL-1β, IL-6, IL-8 levels were significantly positive correlation, r=0.516, P<0.001; r=0.471, P<0.001; r=0.442, P<0.001; r=0.378, P<0.01, while IL-10 levels were negatively correlated, r=-0.319, P<0.01.4. Conclusion(1) miRNA-146a expression can be detected in chronic periodontitis and healthy, and the former expression was significantly higher than the latter.(2) In chronic periodontitis, with increasing degree of periodontal destruction, miRNA-146a expression in supernatant saliva tended to increase.(3) miRNA-146a expression in the chronic periodontitis group had a positively correlation with gingival index, probing depth, attachment loss and plaque index, which suggest that it is closely related to the degree of periodontal destruction.(4) miRNA-146a expression in patients with chronic periodontitis had a positively correlation with the levels of TNF-a, IL-1β, IL-6, IL-8, while had a negative correlation with IL-10PartⅡ The impact of periodontal therapy on miRNA-146a expression and related factors in salivary of patients with chronic periodontitis1. ObjectUsing stem-loop real-time PCR to detect miRNA-146a expression before and after basic periodontal treatment in the supernatant of patients with chronic periodontitis, and analyze the changing trends of miRNA-146a expression before and after the treatment, as well as the relationship with the degree of chronic pariodontis, in order to investigate whether miRNA-146a in supernatant can be the efficacy objective for the monitoring of treatment.2. Methods64 cases chronic periodontitis had basis of periodontal treatment for the study, and had oral hygiene instruction before treatment including the frequency, method of toothbrush, flossing, etc. Then underwent ultrasound on full-mouth scaling gingival surgery, scaling and root planing subgingival, and whole process was completed in two weeks. Not given antibiotics adjuvant therapy after treatment.6th weekend patients were asked to recheck periodontal clinical parameters, miRNA-146a expression and cytokines TNF-a, IL-1β, IL-6, IL-8 and IL-10 levels in the supernatant. Using SPSS 17.0 statistical software to analyze results. Measurement data were showed by x±s, and miRNA-146a expression levels before and after treatment were compared by paired t test, with P<0.05 considering statistically significant. The relationship among miRNA-146a change value (AmiRNA-146a) before and after treatment, periodontal clinical indicators and cytokine changes value were analyzed by the Pearson correlation coefficient.3. Result(1) At last 48 cases completed the basic treatment and referral as required. After six weeks of treatment, observed that gingival index, probing depth, attachment loss and plaque index were significantly lower than before treatment, the difference was statistically significant (P<0.01).(2) Before treatment miRNA-146a expression of the 48 cases (baseline) was 2.85±0.92, after six weeks treatment was 1.49±0.52, the difference was statistically significant (P<0.05).(3) Line correlation analysis displayed, before and after the treatment, changes of miRNA-146a (AmiRNA-146a) had a significant positive correlation with the changes of gingival index (ΔGI), probing depth (ΔPD), attachment loss (AAL) and plaque index (ΔPLI). r=0.412, P<0.01; r=0.506, P<0.001; r=0.470, P<0.001; r=0.584, P< 0.001.(4) After six weeks of treatment TNF-a, IL-1β, IL-6 and IL-8 expression were significantly decreased compared with before treatment (baseline), the difference was statistically significant.(5) Before and after treatment, changes of miRNA-146a (ΔmiRNA-146a) had with a significant positive correlationship with the alternation of TNF-a, IL-1β, IL-6, IL-8(ATNF-a, AIL-1β, AIL-6, AIL-8). r=0.547, P<0.001; r=0.416, P<0.001; r=0.352, P<0.01; r=0.327, P<0.01, while had a negative correlation with changes of IL-10 in the value (AIL-10), r=-0.403, P<0.01.4. Conclusion(1) Basic periodontal treatment can significantly reduce plaque index, gingival index, probing depth and attachment loss index of chronic periodontitis, suggesting that basic treatment can effectively improve periodontal inflammatory conditions.(2)After basic periodontal treatment, miRNA-146a expression in the supernatant of chronic periodontitis patients decreases.(3) miRNA-146a changes had a positive correlation with the alternation of gingival index, probing depth, attachment loss and plaque index before and after basic periodontal therapy.(4) miRNA-146a changes had a positive correlation with the alternation of TNF-a、IL-1β、IL-6、IL-8 in supernatant before and after treatment, while had a negative correlation with IL-10.