MiR-1a-3p Inhibits Apoptosis in Fluoride-exposed LS8 Cells by Targeting Map3k1.

Dental fluorosis is a common chemical disease. It is currently unclear how fluorosis occurs at the molecular level. We used miRNA-seq to look at the differences between miRNAs in the cell line of ameloblasts LS8 that had been treated with 3.2 mmol/L NaF. We also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. miR-1a-3p levels were significantly lower in mouse LS8 cells treated with 3.2 mmol/L NaF, and miR-1a-3p-targeted genes were significantly enriched in the MAPK pathway. LS8 cells were divided into four groups: control, NaF, NaF+miR-1a-3p mimics, and NaF+miR-1a-3p mimics normal control groups. Cellular morphology was observed by an inverted microscope, and the proliferation activity of LS8 cells was assessed by Cell Counting Kit-8 (CCK-8). Using the real-time quantitative polymerase chain reaction (RT-qPCR), transcription levels of miR-1a-3p and Map3k1 were detected. The expressions of Bax, Bcl-2, Map3k1, p38MAPK, ERK1/2, p-p38MAPK, and p-ERK1/2 were measured by Western blot. After bioinformatics analysis, we used a luciferase reporter assay (LRA) to validate the target of miR-1a-3p, showing that miR-1a-3p could inhibit apoptosis while increasing proliferation in fluoride-exposed LS8 cells. Generally, miR-1a-3p might directly inhibit Map3k1, reduce MAPK signal pathway activation, and promote phosphorylation. Thus, our findings revealed that the interaction of miR-1a-3p with its target gene Map3k1 and MAPK signal pathway might decrease the apoptosis of LS8 cells treated with 3.2 mmol/L NaF.

Effects of epigallocatechin-3-gallate on oxidative stress, inflammation, and bone loss in a rat periodontitis model.

Background/purpose: Epigallocatechin-3-gallate (EGCG) is playing an increasingly important role in the treatment of oral diseases. However, its mechanisms remain to be clarified. This study aimed to investigate the effect of EGCG on oxidative and inflammatory stress and bone loss in experimental periodontitis. Materials and methods: Periodontitis was induced in rats, followed by gavage using different concentrations of EGCG for 5 weeks. The levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD) and malondialdehyde (MDA) in rats were measured. The degree of alveolar bone loss and the number of inflammatory cells were detected. The integrated optical density of nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), NLR pyrin domain-containing 3 (NLRP3) and nuclear factor-kappaB p65 (NF-κB p65) was measured. Results: EGCG (200 mg/kg) significantly reduced alveolar bone loss in the ligated maxillary molars and the number of inflammatory cells in the EGCG-200 group compared with the periodontitis, EGCG-100 and EGCG-400 groups. 200 mg/kg was the optimal dose of EGCG and was used in subsequent experiments. The expression levels of IL-1ß, IL-18, TNF-α and MDA were significantly lower and the expression level of SOD was significantly higher in the EGCG-200 group compared with the periodontitis group. The expression of NLRP3 and NF-κB p65 was significantly decreased, while the expression of Nrf2 and HO-1 was significantly increased in the EGCG-200 group compared with the periodontitis group. Conclusion: These results suggest that EGCG inhibits oxidative stress and inflammatory responses in the periodontitis model by modulating the Nrf2/HO-1/NLRP3/NF-κB p65 signaling pathway, thereby decreasing alveolar bone loss.

Development of an miRNA-Array-Based Diagnostic Signature for Periodontitis.

Periodontitis progression is accompanied by irreversible alveolar bone absorption and leads to tooth loss. Early diagnosis is important for tooth stability and periodontal tissue preservation. However, there is no recognized miRNA diagnostic signature with convincing sensitivity and specificity for periodontitis. In this study, we obtained miRNA array expression profiles of periodontitis from the Gene Expression Omnibus (GEO) database. After screening for differentially expressed miRNAs, the least absolute shrinkage and selection operator (LASSO) method was performed to identify and construct a 17-miRNA-based diagnostic signature (hsa-miR-3917, hsa-mir-4271, hsa-miR-3156, hsa-miR-3141, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-671-5p, hcmv-mir-UL70, hsa-miR-650, hsa-miR-497-3p, hsa-miR-145-3p, hsa-miR-141-3p, hsa-miR-210-3p, hsa-miR-204-3p, hsa-miR-203a-5p, hsa-miR-99a-3p, and hsa-miR-30a-3p). Periodontal tissue samples with higher risk scores were more likely to show symptoms of periodontitis. Then, the receiver operating characteristic (ROC) curves were used to assess the diagnostic value of the miRNA signature, which indicated that the optimum cutoff value in periodontitis diagnosis was 0.5056 with an area under the ROC curve (AUC) of 0.996, a sensitivity of 97.3%, a specificity of 100.0% in the training cohort; in the testing cohort, the corresponding values were as follows: an AUC of 0.998, a sensitivity of 97.9%, and a specificity of 91.7%. We next evaluated the efficacy of the signature in differentiating disease subtype and affected range. Furthermore, we conducted functional enrichment analysis of the 17 miRNA-targeted mRNAs, including the regulation of mTOR activity and cell autophagy, Th1/Th2 cell balance and immunoregulation, cell apoptosis, and so on. In summary, our study identified and validated a 17-miRNA diagnostic signature with convincing AUC, sensitivity, and specificity for periodontitis.

TLR4 polymorphism and periodontitis susceptibility: A meta-analysis.

BACKGROUND: Many primary and secondary studies reported the association between Toll-like receptor 4 (TLR4) polymorphism and periodontitis susceptibility, which mainly focused on TLR4-299A>G or TLR4-399C>T of Caucasian, however, these studies had different conclusions. The aim of this study was to reassess relative studies about TLR4 polymorphism and periodontitis susceptibility, and update meta-analysis. METHODS: We searched the electronic database including CNKI (Chinese National Knowledge Infrastructure), PubMed, Embase, and hand searched relative studies until January 4, 2016. Two authors selected studies according to inclusion and exclusion criteria, assessed studies using Newcastle-Ottawa Scale case control study (NOS), and calculated the combined effect size using STATA software, version 12.0. RESULTS: This meta-analysis included 18 studies, containing 2453 healthy participants and 2987 patients with chronic periodontitis (CP) and 462 patients with aggressive periodontitis (AP). There was a significance between TLR4C>G (rs7873784) allele and CP in Asian, and its recessive model was also significant (for C vs G: odds ratio [OR] = 0.72, 95% confidence interval [CI] = 0.54-0.95, I = 0%; for CC + CG vs GG: OR = 0.66, 95% CI = 0.49-0.89, I = 0%). However, we did not detect any significant relevance between other TLR4 polymorphism and periodontitis susceptibility in overall and subgroup analyses. The sensitive analysis showed that dropping any single studies did not affect the pooled-analysis results. Publication bias was not detected. CONCLUSIONS: The meta-analysis found association between TLR4C>G (rs7873784) allele and CP in Asian and it may passed on to offsprings in the form of recessiveness. However, further studies about the association between TLR4C>G (rs7873784) and CP is warranted to confirm.

[Expression of Streptococcus mutans surface protein PAcP and cholera toxin B subunit fusion gene in transgenic tomato].

PURPOSE: To detect the protein expression level of foreign fused gene in the first generation of genetically modified tomatoes transformed with fused gene of antigen epitope PacP in surface protein of Streptococcus mutans and cholera toxin B subunit. METHODS: The total DNA in tomatoes was extracted and PCR was applied to screen the first generation of transgenic tomatoes carrying Streptococcus mutans surface protein coding PAcP with cholera toxin B subunit fusion gene. Total proteins were extracted and quantitatively tested with BAC kit. Foreign fused protein expression level was analysed by Western blotting and ELISA. RESULTS: Ten DNA samples with the full length of 1.6 kb foreign fused DNA fragment was detected by PCR among all 18 samples. The crude protein in the transgenic tomato fruit tissue was 3.93 mg/mL. Compared with the normal tomatoes, Western blotting showed that the transgenic tomato protein contained a distinct protein band with a molecular weight about 60 kD. The results of ELISA suggested that fused foreign protein level was up to 0.18% of the total soluble protein in genetically modified tomato fruit. CONCLUSIONS: The target protein encoded by fused foreign gene of antigen epitope PacP in surface protein and cholera toxin B subunit was expressed in genetically modified tomatoes fruit.

[Study on gene vaccine pcDNA3-PAc against dental caries by intranasal immunization in rabbits].

PURPOSE: To investigate the efficiency of pcDNA3-PAc on Japanese long-eared white rabbits by intranasal immunization, and observe the appreciate gene vaccine dose in rabbit immunization. METHODS: Thirty Japanese long-eared white rabbits were randomly divided into 5 groups (6 in each group) as follows: 200, 400, 600 µg pcDNA3-PAc plasmid group; 400 µg pcDNA3 group and inactivated whole-cell vaccine positive control group. The rabbits were immunized twice, and plasmid groups and whole-cell group were coupled Freund's adjuvant with 1:1 ratio. The specific IgG and S-IgA antibodies in serum and saliva were detected with indirect ELISA method. The data was analyzed with SPSS 17.0 software package. RESULTS: (1) The peak time of the antibodies appeared between 8-10 weeks after the first immunization; (2)The specific antibodies IgG and S-IgA could be detected 2 weeks after immunization;(3)The level of salivary specific S-IgA and serum specific IgG of pcDNA3-PAc were significantly higher than negative groups (P<0.05). The titer of the 200 µg was significantly lower than those of 400 µg and 600 µg group (P<0.05). CONCLUSIONS: (1)The recombinant plasmid pcDNA3-PAc has immunogenicity, which can induce specific immune responses for 14 weeks in rabbits. (2)The results of the present study show that 200 µg, 400 µg and 600 µg are effective immunizational dosage to 1.5 kg Japanese long-eared white rabbits. (3)400 µg and 600 µg pcDNA3-PAc can be considered as the optimal dosage than 200 µg at present experimental condition. Supported by Guizhou Science and Technology Projet of Distinguished Young Talents [2005(0509)], Guizhou College and University Leading Academic Discipline Project [2012(15)] and Zunyi Medical University Project of Distinguished Research Team [2012(12)].

[Detection of the exogenous gene copy number of the transgenic tomato anti-caries vaccine].

PURPOSE: To detect the exogenous gene copy number of the transgenic tomato anti-caries vaccine by using the SYBR Green real-time PCR. METHODS: Recombinant plasmid pEAC10 and pEPC10 were used as standard to detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR green fluorescent quantitation, then the average value was calculated as gene copy number. RESULTS: The copy number of the transgenic tomato carrying pacA-ctxB was 1.3 and the pacP-ctxB was 3.2. CONCLUSIONS: The transgenic tomato plants which have high stability are low-copy transgenic plants. Supported by National Natural Science Foundation of China (30160086, 81260164), Science and Technical Fund of Guizhou Province (LKZ[2011]41), Project of Technology Innovation Team in Guizhou Province, Leading Academic Discipline Construction Project in Guizhou Province and Excellent Scientific Research Team Cultivation Project in Zunyi Medical College ([2012]12).