Identification of novel key lncRNAs involved in periodontitis by weighted gene co-expression network analysis.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial disease that can lead to the progressive destruction of dental support tissue. However, the detailed mechanisms and specific biomarkers involved in periodontitis remain to be further studied. Recently, long non-coding RNAs (lncRNAs) have been found to play a more important role than other types of RNAs. In our study, we analysed the expression of lncRNAs in periodontitis by analysing GSE16134. MATERIAL AND METHODS: We identified highly correlated genes by analysing GSE16134 with weighted gene co-expression network analysis (WGCNA) and identified 50 hub lncRNAs that were dysregulated. Then, we used the Linear Models for Microarray Data (Limma) package to identify the hub lncRNAs that were differentially expressed (DElncRNAs). The ceRNA co-expression network data were obtained from several sites, including miRcode, and were used to assess the potential WGCNA function of hub DElncRNAs in periodontitis. Besides, we divided the samples into LBX2-AS1 high and low expression group by the expression level of LBX2-AS1 and calculated DEG by Limma package. Furthermore, we performed GO function, KEGG pathway and GSEA enrichment of DEGs. RESULTS: In the analysis, we identified 50 hub lncRNAs that may play important roles in periodontitis. Then, we used the Limma package to identify 3 hub DElncRNAs (LINC00687, LBX2-AS1 and LINC01566). We elucidated the potential function of the hub DElncRNA LBX2-AS1 in periodontitis by constructing a co-expression network of lncRNA-miRNA-mRNA interactions. Totally, 573 DEGs (354 up- and 219 downregulated) in periodontitis samples were identified. DEGs were enriched in different GO terms and pathways, such as neutrophil degranulation, neutrophil activation, neutrophil activation involved in immune response, neutrophil-mediated immunity, antigen processing and presentation, JAK-STAT signalling pathway, natural killer cell-mediated cytotoxicity, EGFR tyrosine kinase inhibitor resistance, phosphatidylinositol signalling system and Vascular Endothelial Growth Factor (VEGF) signalling pathway. CONCLUSION: In our study, we found that 3 hub DElncRNAs (LINC00687, LBX2-AS1 and LINC01566) may be involved in the pathogenesis of periodontitis based on WGCNA and Limma analysis. Our study aimed to elucidate the mechanisms involved in periodontitis at the genetic and epigenetic levels by constructing a ceRNA network associated with lncRNA. Besides, identification DEGs of differential LBX2-AS1 and functional annotation showed that LBX2-AS1 might be associated with periodontitis.

[Experimental study of APB-DOCK8 transgenic tomato vaccine for caries prevention].

PURPOSE: To observe the anti-caries effect of transgenic tomato anti-caries vaccine after immunization with SD rats by gavage and to explore its immunity mechanism initially. METHODS: SD rats were used to establish an experimental caries model. The transgenic anti-caries tomatoes expressing the target protein were cultivated and identified. The SIgA and IgG contents of specific anti-PAcA in saliva and blood samples of SD rats were detected by ELISA. Then, the SD rats were sacrificed, the maxillary and mandibular bones were taken for Keyes dental caries score, and spleens were taken for the analysis of RNA-seq. Statistical analysis was performed with SPSS 18.0 software package. RESULTS: The target protein concentration in the transgenic tomato anti-caries vaccine was 36.28 µg/mL. After vaccine immunization of SD rats, group D (8 mL/kg) produced the highest levels of specific SIgA and IgG antibodies at week 6 and was significantly different from the other groups(P＜0.05), and caries counting score was also significantly different than the other groups (P＜0.05). The spleen mRNA of SD rats in group D was extracted and sequenced by RNA-seq, and 40 genes with significant differences in mRNA expression were obtained(P-adjust＜0.05, |Fold Change|≥1.5). 26 genes were significantly upregulated, including IGFBP6 and COL15A1. The upregulated gene GO enrichment was enriched to humoral immune response, B-cell activation, and immunoglobulin receptor binding; KEGG enrichment was enriched to 56 signaling pathways, including PI3K-AKT and NF-κB, and F＜0.001. Fourteen genes were significantly downregulated, but the analysis of downregulated gene GO and KEGG enrichment was not statistically significant(F＞0.1). CONCLUSIONS: Transgenic tomato anti-caries vaccine may reduce caries occurrence by upregulating the activation of PI3K-AKT signaling pathway mediated by IGFBP6 in SD rats.

Identification of immunological bioprocesses involved in peri-implantitis using weighted gene co-expression network analysis.

BACKGROUND: Peri-implantitis is an irreversible infectious disease that occurs with high incidence. Exploring the immune responses of peri-implantitis is key to developing targeted treatment strategies. However, there is limited research on the immune response of peri-implantitis. METHODS: This study performed a weighted gene co-expression network analysis to identify the peri-implantitis related gene network and conducted a functional enrichment analysis of the gene network. Thereafter, the candidate hub genes were selected by constructing a protein-protein interaction network and drawing an upset plot. The hub genes were identified through their significant associations with disease condition and validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Using the gene set variation analysis, the hub genes were further used to explore infiltrating immunocytes and immune factors in peri-implantitis. Finally, the immunocytes and immune factor related hub genes were intersected to obtain the therapeutic target, which was validated using histological staining. RESULTS: The peri-implantitis related gene network was enriched in innate and adaptive immune response. Subsequently, interleukin (IL)1B, IL10, ITGAM, ITGB1, STAT3, and TLR4 were identified as hub genes. Plasmacytoid dendritic cells, macrophages, myeloid-derived suppressor cells, natural killer T cells, and immature B cells were positively and significantly related to the hub genes IL1B, TLR4, ITGAM, and ITGB1 (correlation coefficient > 0.80). While immune factors CXCL10, IL6, and CXCL12 and hub genes IL10 and IL1B held the highest degree in the immune factors network. IL1B may be a promising therapeutic target. CONCLUSION: This study provides new insights into the hub genes, immunocytes, and immune factors underlying peri-implantitis immunological bioprocess.

Recombinant human insulin-like growth factor-1 promotes osteoclast formation and accelerates orthodontic tooth movement in rats.

BACKGROUND: IGF-1 may be an important factor in bone remodeling, but its mechanism of action on osteoclasts during orthodontic tooth movement is complex and unclear. METHODOLOGY: The closed-coil spring was placed between the left maxillary first molar and upper incisors with a force of 50 g to establish an orthodontic movement model. Eighty SD rats were randomized to receive phosphate buffer saline or 400 ng rhIGF-1 in the lateral buccal mucosa of the left maxillary first molar every two days. Tissue sections were stained for tartrate-resistant acidic phosphatase (TRAP), the number of TRAP-positive cells was estimated and tooth movement measured. RESULTS: The rhIGF-1 group exhibited evidential bone resorption and lacuna appeared on the alveolar bone compared to the control group. Moreover, the number of osteoclasts in compression side of the periodontal ligament in the rhIGF-1 group peaked at day 4 (11.37±0.95 compared to 5.28±0.47 in the control group) after the orthodontic force was applied and was significantly higher than that of the control group (p<0.01). Furthermore, the distance of tooth movement in the rhIGF-1 group was significantly larger than that of the control group from day 4 to day 14 (p<0.01), suggesting that rhIGF-1 accelerated orthodontic tooth movement. CONCLUSION: Our study has showed that rhIGF-1 could stimulate the formation of osteoclasts in the periodontal ligament, and accelerate bone remodeling and orthodontic tooth movement.

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.

Recombinant human insulin-like growth factor-1 promotes osteoclast formation and accelerates orthodontic tooth movement in rats

Abstract Background: IGF-1 may be an important factor in bone remodeling, but its mechanism of action on osteoclasts during orthodontic tooth movement is complex and unclear. Methodology: The closed-coil spring was placed between the left maxillary first molar and upper incisors with a force of 50 g to establish an orthodontic movement model. Eighty SD rats were randomized to receive phosphate buffer saline or 400 ng rhIGF-1 in the lateral buccal mucosa of the left maxillary first molar every two days. Tissue sections were stained for tartrate-resistant acidic phosphatase (TRAP), the number of TRAP-positive cells was estimated and tooth movement measured. Results: The rhIGF-1 group exhibited evidential bone resorption and lacuna appeared on the alveolar bone compared to the control group. Moreover, the number of osteoclasts in compression side of the periodontal ligament in the rhIGF-1 group peaked at day 4 (11.37±0.95 compared to 5.28±0.47 in the control group) after the orthodontic force was applied and was significantly higher than that of the control group (p<0.01). Furthermore, the distance of tooth movement in the rhIGF-1 group was significantly larger than that of the control group from day 4 to day 14 (p<0.01), suggesting that rhIGF-1 accelerated orthodontic tooth movement. Conclusion: Our study has showed that rhIGF-1 could stimulate the formation of osteoclasts in the periodontal ligament, and accelerate bone remodeling and orthodontic tooth movement.

[Effect of local injection of rhTGF-α1 on osteoclasts during orthodontic tooth movement in rats].

PURPOSE: To investigate the effect of exogenous TGF-ß1 on improving periodontal tissues remodeling during orthodontic tooth movement in rats. METHODS: Eighty male SD rats were randomly divided into 2 groups. Rats in the experimental group were injected respectively with rhTGF-ß1 in dosages of 0.1 mL (5 ng/mL) in the buccal submucosal area of the molar every other day, while rats in the control group received equivalent volumes of PBS. Each group (n=40) was subdivided equally into 5 subgroups. An orthodontic appliance, consisting of a 5 mm nickel titanium closed coil spring, was ligated between the maxillary left incisor and first molar of each rat to deliver an initial force of 0.49N. Eight rats in each subgroup were sacrificed at one of the five time points (1, 4, 7, 10 and 14 days) after appliance placement. The distance of the tooth movement was measured by using stereomicroscope. Tissue sections around the first maxillary left molar were stained with tartrate-resistant acid phosphatase (TRAP) histochemistry to analyze the changes of the amount and distribution of osteoclasts on the compression side of tooth. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Molars treated with rhTGF-ß1 moved mesially more rapidly than the control group. The distance of tooth movement of the experimental group showed a significant increase compared with the control group at day 7 (P<0.05) and significant increase compared with the control group at day 10 and 14 (P<0.01). The number of TRAP positive cells appearing in the periodontal ligament space on the pressure side of the alveolar bone were increased markedly in experimental group. There were statistically significant differences in the amount of osteoclasts between experimental and control groups (P<0.01). CONCLUSIONS: Local injection of rhTGF-ß1 in the periodontal tissues can accelerate orthodontic tooth movement via enhancing the numbers of osteoclasts. At the histologic level, increased numbers of mononuclear osteoclasts are recruited and activated, resulting in greater amounts of alveolar bone resorption on the pressure side of the periodontal ligament.

[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)].

[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.

[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).

Immune responses on allograft heart transplantation in inbred rats infected with Echinococcosis multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis (E. multilocularis) and is a rare but life-threatening disease. This disease commonly is characterized by an infiltrative, tumor-like growth of the E. multilocularis metacestode in the liver of human. Liver transplantation is an effective therapy for end-stage of hepatic AE, but the characteristics of host immunity associated with E. multilocularis infection with organ transplantation are poorly defined. We hereby aimed to study the immunological status and allograft heart survival in inbred rats with E. multilocularis infection. METHODS: Rat models of AE were established by injecting the E. multilocularis suspension made from E. multilocularis infected tissues into the abdomen of Lewis (LEW) rats. Three months later, in the experimental group, allograft heart transplantation was performed from Brown-Norway (BN) rats to the E. multilocularis infected LEW rats. In the control group, we transplanted hearts from BN rats to healthy LEW rats. The influence of the disturbed immune system in E. multilocularis infected rats on the heart transplantation was assessed, including observation of allograft heart survival time, histopathological examination of grafts and immunohistochemical examination of infiltrating cells (CD4(+) T cells, CD8(+) T cells and eosinophile granulocytes), measurement of interleukin (IL)-4 and interferon (IFN)-γ in the serum by enzyme-linked immunosorbent assay (ELISA) and analysis of CD4(+)CD25(+) regulatory T cells in peripheral blood by fluorescence activated cell sorting (FACS) flow cytometric analysis. RESULTS: The survival time of recipients in the experimental group was prolonged compared with those in the control group. The numbers of graft infiltrating CD8(+) T cells were decreased whereas the graft infiltrating eosinophil granulocytes (CD15(+)) were increased in grafts in the experimental group (P < 0.05). Furthermore, the proportion of CD4(+)CD25(+) regulatory T cells in the peripheral blood was 10.8% on average in the experimental group, which was significantly higher than that in the control group (6.1%). In addition, the level of serum IL-4 in E. multilocularis infected rats was higher than that in the control group rats, whereas the level of serum IFN-γ in experimental group was lower than that in the control group when graft rejection occurred (P < 0.05). CONCLUSIONS: This study suggests that E. multilocularis infection could prolong the allograft survival time through the polarization of Th1/Th2-type cells and induction of CD4(+)CD25(+) regulatory T cells. This strategy may provide a new idea for establishing transplantation tolerance.

[Isolation and characterization of a phenanthrene-degrading bacterial strain].

A bacterial strain that degrades phenanthrene was isolated from activated sludge of wastewater treatment plant from a chemical corporation. The strain can utilize phenanthrene as sole source of carbon and energy. It was identified as Burkholderia sp. according to morphological and 16S rRNA gene sequence analysis. Phenanthrene (100 mg x L(-1)) was removed 74.1% within 84 h by this strain. It was suggested that the strain possessed the salicylic acid pathway by LC-MS and UV methods. And the strain contains an inductive catechol 2,3-dioxygenase (C230). The kinetic constants Km and Vmax were 10.93 micromol x L(-1) and 106.38 micromol x min(-1), receptively. It indicates that the affinity of the endoenzyme to catechol is great and catechol is a better substrate.