Possible salivary and serum biomarkers for oral lichen planus

There are few reports concerning the potential for clinical application of oxidative stress (OS) and collagen degradation markers in oral lichen planus (OLP) patients. We investigated the possibility of using some disease-related biomarkers in saliva and serum of OLP patients. Our study included 30 patients with OLP and 30 controls. We evaluated serum and salivary OS biomarkers including 8-OHdG, MDA, uric acid, TAC and GPx. We also investigated collagen degradation markers such as CTX I and MMP-8. We found significantly increased salivary levels of MMP-8 and CTX I in the OLP group compared to controls and significant differences between the OLP and control groups in serum and saliva for 8-OHdG, MDA (significantly increased), uric acid, TAC and GPx (significantly reduced). Currently there are no criteria for evaluating which OLP patients have a greater risk of malignant transformation. In addition to clinical surveillance, the serum and salivary biomarkers that we evaluated may be useful biomarkers for monitoring OLP patients in the future.     

Cytometric analysis of oral scrapings of patients with oral lichen planus

A. Acha‐Sagredo, Y. Jiménez, J. V. Bagán, M. A. Echebarria‐Goicouria and J. M. Aguirre‐Utric analysis of oral scrapings of patients with oral lichen planus Objective: Aneuploidy has been associated with malignant and premalignant oral lesions. In the past few years, its application in oral precancerous lesions and its prognostic meaning have been controversial issues. The aim of our study was to characterize alterations in DNA content by automated DNA image cytometry in oral scrapings of patients with oral lichen planus. Methods: Cytological samples from 40 patients clinicopathologically diagnosed with oral lichen planus were analysed by DNA image cytometry. Results: All the cases were classified as diploid, showing a predominant population of cells with normal DNA content (DNA index, 0.85–1.15). Atrophic/erosive lesions showed a higher percentage of tetraploid cells when compared with reticular/papular lesions but this was not statistically significant (P = 0.09). Conclusions: Aneuploidy does not seem a common event in oral lichen planus lesions. However, we consider that the use of DNA image cytometry of oral scrapings may be an easy and helpful methodology in the follow‐up of patients with these lesions.     

Phenotypic variability and therapeutic implications of Candida species in patients with oral lichen planus

We investigated the prevalence and phenotypic variation of Candida species in oral lichen planus (OLP) and the therapeutic implications of our findings. Eighty patients with clinically and histopathologically confirmed cases of OLP (64 non-erosive, 16 erosive) and a control group of 80 healthy individuals with no predisposing factors for oral candidiasis were examined for evidence of Candida infection. Oral swabs and smears were obtained for cytology and culture. Identification, speciation and antifungal susceptibility tests of Candida isolates were performed using an automated microbial identification system. Fifty percent of erosive OLP cases, 28% of non-erosive cases and none of the controls showed evidence of Candida. Candida albicans was found predominantly in non-erosive OLP, while other Candida species were predominate in erosive OLP. Non-Candida albicans isolates (C. glabrata, C. krusei) were resistant to the commonly used antifungals, clotrimazole and fluconazole. Candida infection is common in cases of OLP. We recommend antifungal sensitivity testing prior to antifungal therapy for the erosive form of OLP.     

Detection and identification of non-Candida albicans species in human oral lichen planus

Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non-C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non-C. albicans are specifically present in subjects with this condition.     

Alteration of salivary glycopatterns in oral lichen planus

Background: Oral lichen planus (OLP) is one of the most common oral mucosal lesions affecting 0.5–2% of the adult population. It is difficult to distinguish between OLP and other oral mucosal diseases. Structural changes in the glycans of saliva proteins might be reliable indicators of OLP. However, little is known about the alteration of salivary glycopatterns during OLP.

Objective: We aimed to investigate the alterations of salivary protein glycosylation related to OLP.

Material and methods: Twenty-eight patients with OLP and 30 age- and sex-matched healthy volunteers (HVs) were enrolled in the test group to probe the difference of salivary glycopatterns using lectin microarrays. The lectin blotting were further utilized to validate the expression of certain glycans.

Results: The glycoproteins recognized by three lectins [Aleuria aurantia lectin (AAL); Phytolacca americana (PWM); Phaseolus vulgaris agglutinin (E?+?L), (PHA-E?+?L)] were mainly increasing in the saliva of OLP. Meanwhile, these glycoproteins also exhibited significant age-associated alterations.

Conclusions: This study provided a new basic insight into salivary glycopatterns in OLP and helped to develop new potential biomarkers for diagnosis of OLP.     

Relationship between human oral lichen planus and oral squamous cell carcinoma at a genomic level: a datamining study

The leader gene approach is a data mining method based on the systematic search for genes involved in a specific process and their ranking according to the number of interconnections with the other genes identified. The genes with the strongest interconnections are termed leader genes, since they may be supposed to play an important role in the process. The potential of malignant progression of OLP to oral squamous cell carcinoma (OSCC) is still not completely clear. In this study, the leader gene approach is applied to investigate the association between OLP and OSCC at a molecular level. Results were integrated with those obtained in an experimental analysis (see paper 1 of this series). Genes involved in OLP and OSCC were identified by systematic queries to dedicated databases. Interconnections among identified genes were calculated and given a confidence value using STRING database. Leader genes were identified by clustering genes according to their interconnections. This theoretical analysis shows that OLP and OSCC share two leader genes: TP53 and CDKN1A, involved in the PI3K signalling events mediated by AKT pathway. This finding and those obtained in the experimental analysis suggest the possible involvement of some key genes/proteins LCK, PIK3CA, BIRC5, TP53 and CDKN1A in the malignant progression from OLP to OSCC. Moreover, these findings support the role of some molecular pathways, namely IL2 signalling events mediated by PI3K, PI3K signalling events mediated by AKT, and, possibly, Aurora A signalling in the association between OLP and OSCC.     

Analysis of oral microbial community and Th17‐associated cytokines in saliva of patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa of unknown cause. Microbial infection and dysimmunity appear to play important roles in its pathogenesis. In this study, differences in genetic profiling of salivary microbial communities in two subtypes of OLP and healthy controls were evaluated by means of PCR‐denaturing gradient gel electrophoresis (DGGE). Additionally, ELISA was used to investigate the possible role of Th17 in lesion formation by detecting two related cytokines IL‐17 and IL‐23 in the saliva of OLP patients. When the DGGE profiles were analyzed, the bacterial populations were found to be significantly less rich in subjects with reticular and erosive OLP than in healthy controls. There was significantly less microbial diversity, as denoted by the Shannon index, in saliva samples from subjects with erosive OLP than in those from healthy controls. Cluster analysis and principal component analysis showed that the DGGE profiles formed distinctly group‐specific clusters. Salivary concentrations of IL‐17 in subjects with erosive OLP group were significantly higher than in those with reticular OLP and healthy controls. What's more, significantly positive correlations were observed between salivary IL‐17 concentrations and disease clinical scores. Microbial richness and diversity was negatively correlated with salivary IL‐17 concentrations. These results suggest there is significantly less salivary bacterial diversity and complexity in subjects with OLP han in healthy controls and that the shifted community composition is closely related to an immune cytokine, IL‐17.     

Candida biotypes in patients with oral leukoplakia and lichen planus

Prevalence of yeasts in 35 leukoplakia and 34 oral lichen planus patients was compared with that observed in persons without oral diseases. Serotype and morphotype were determined on Candida albicans isolates. Yeasts were isolated from the oral cavity specimens of 43.7% of the patients. C. albicans (serotype A) was the predominant species (76% in leukoplakia, 88.2% in lichen planus and 60.8% in healthy persons). Sixteen morphotypes were encountered on malt extract agar, being 732, 733, 734, 753 and 754 the most frequently found. Morphotypes SP1N and SP1Y were the most common on Sabouraud-trypheniltetrazolium agar (68.4% of the isolates from leukoplakia and 73.3% from lichen planus, but only 46.6% of the isolates from healthy oral mucosa showed SP1N morphotype). Presence of oral lesions was associated with a marked reduction in the yeast species and C. albicans biotypes, suggesting that C. albicans and particularly some of its biotypes, show a high potential of adaptation to the changes associated with the development of oral leukoplakia and lichen planus.     

Metabolic changes during malignant transformation in primary cells of oral lichen planus: Succinate accumulation and tumour suppression

Oral squamous cell carcinoma (OSCC) is usually diagnosed at late stages, which leads to high morbidity. There are evidence that chronic inflammation (eg oral lichen planus [OLP]) was a risk factor of OSCC, but often misdiagnosed or ignored until invasion and metastasis. By applying precision medicine, the molecular microenvironment variations and relevant biomarkers for the malignant transformation from OLP to OSCC can be fully investigated. Several studies pointed out that the metabolic pathway were suppressed in OSCC. However, it remains unclear how the systemic profile of the metabolites change during the malignant transformation. In this study, we examined and compared the mucosa samples from 11 healthy individuals, 10 OLP patients and 21 OSCC patients. Based on the results, succinate, a key metabolite of the tricarboxylic acid cycle pathway, was accumulated in the primary cultured precancerous OLP keratinocytes and OSCC cells. Then, we found that succinate activated the hypoxia‐inducible factor‐1 alpha (HIF‐1α) pathway and induced apoptosis, which could also be up‐regulated by the tumour suppressor lncRNA MEG3. These results suggested the critical roles of succinate and MEG3 in the metabolic changes during malignant transformation from OLP to OSCC, which indicated that succinate, HIF1α and downstream proteins might serve as new biomarkers of precancerous OLP for early diagnosis and therapeutic monitoring. In addition, succinate or its prodrugs might become a potential therapy for the prevention or treatment of OSCC.     

细胞免疫功能及辅助型T细胞因子在口腔扁平苔藓中的作用

目的 探讨细胞免疫功能及辅助型T细胞（Th）细胞因子在口腔扁平苔藓（OLP）中的作用。方法 以35例糜烂型（糜烂组）及29例网纹型OLP（网纹组）患者为对象,选取同期20例健康人为对照组,流式细胞术检测外周血中T细胞、B细胞、NK细胞和调节性T细胞亚群表达百分率。ELISA法检测外周血中TNF-α和INF-γ（Th1型细胞因子）,IL-4和IL-10（Th2型细胞因子）,IL-17、IL-23（Th17型细胞因子）含量;分离外周血单个核细胞,qRT-PCR检测上述因子mRNA的表达。结果 与健康组相比,OLP患者CD3+、CD4+ T细胞亚群比例（F=3.211和3.565,P<0.05）及CD4+/CD8+降低（F=3.430,P<0.05）,CD4+Foxp3+调节性T细胞亚群比例（F=3.370,P<0.05）及外周血中TNF-α、INF-γ、IL-4、IL-10、IL-17、IL-23含量增高（F=0.923、0.820、1.043、1.132、0.745和0.802,P<0.05）。与网纹组相比,糜烂组CD8+ T细胞亚群比例较低（t=2.450,P<0.05）;IL-4、IL-10蛋白（t=22.780和25.112,P<0.05）及mRNA较低（t=3.781和6.710,P<0.05）,IL-17、IL-23蛋白（t=15.765和19.307,P<0.05）及mRNA较高（t=4.022和8.569,P<0.05）。结论 口腔扁平苔藓患者存在细胞免疫紊乱及Th细胞因子异常,其中CD8+及Th17型细胞与糜烂型OLP发病有关,Th2型细胞与网纹型OLP发病有关。     

Down‐regulation of miRNA‐27b‐3p suppresses keratinocytes apoptosis in oral lichen planus

Oral lichen planus (OLP) is considered a precancerous lesion with no known cure. Recent studies reported that abnormal regulation of apoptosis was involved in the pathogenesis of OLP. Next generation sequencing was used to screen the candidate microRNAs and genes in biopsies from patients with OLP and healthy mucosa. Human oral keratinocytes were transfected into the related oligonucleotides of miR‐27b‐3p/cyclophilin D and their control groups. Apoptosis was detected by TdT‐mediated dUTP nick end labelling and flow cytometry. The levels of mRNA and protein were detected by quantitative PCR, Western blots, and enzyme‐linked immunosorbent assays, respectively. Luciferase assays were performed to detect the luciferase activities of miR‐27b‐3p and cyclophilin D. Here, we showed that basal epithelium apoptosis was reduced and the miR‐27b‐3p levels were decreased in clinical OLP samples. We also found that down‐regulation of miR‐27b‐3p inhibited epithelial keratinocyte apoptosis by up‐regulating cyclophilin D expression. Moreover, cyclophilin D increased the protein stability of Bcl2 through direct binding, and Bcl2 suppressed caspase9/3 activation and cytochrome C release. Taken together, these data showed that miR‐27b‐3p regulated keratinocyte apoptosis through cyclophilin D/Bcl2 signalling, suggesting the miR‐27b‐3p regulated the pathogenesis of OLP.     

miR-122-5p promotes aggression and epithelial-mesenchymal transition in triple-negative breast cancer by suppressing charged multivesicular body protein 3 through mitogen-activated protein kinase signaling

Triple-negative breast cancer (TNBC) is highly metastatic and frequently has a poor prognosis. The lack of comprehension of TNBC and gene therapy targets has led to limitedly effective treatment for TNBC. This study was conducted to better understand the molecular mechanism behind TNBC progression, and to find out promising gene therapy targets for TNBC. Herein the influence of miR-122-5p's binding charged multivesicular body protein 3 (CHMP3) 3′-untranslated region (3′-UTR) on in TNBC cells was investigated. in vitro experiments quantitative real-time polymerase chain reaction, immunoblot analysis, dual-luciferase reporter gene assay, cell counting assay, transwell invasion assay, and flow cytometry-determined cell apoptosis assay were employed. We also used TargetScan Human 7.2 database to find out the target relationship between miR-122-5p and CHMP3 3′-UTR. TImer algorithm was used to provide an overview of the expression of CHMP3 gene across human pan-cancer, to predict the survival outcome of breast cancer patients, and to predict the correlation between CHMP3 gene expression and epithelial-mesenchymal transition (EMT) and mitogen-activated protein kinase (MAPK)-related gene expression. CHMP3 gene was significantly downregulated across a wide range of human cancers including breast cancer (BRCA). A higher level of CHMP3 gene predicted a better 3- and 5-year survival outcome of patients with BRCA. In our experiments, miR-122-5p was significantly upregulated and CHMP3 gene was significantly downregulated in TNBC cells compared with normal cell line. miR-122-5p mimics enhanced TNBC cell viability, proliferation, and invasion whereas the upregulation of CHMP3 gene led to an opposite outcome. Forced expression of miR-122-5p suppressed cell apoptosis, compelled EMT and MAPK signaling whereas forced expression of CHMP3 did the opposite. We then conclude that miR-122-5p promotes aggression and EMT in TNBC by suppressing CHMP3 through MAPK signaling.     

MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers

Aberrant regulation of APC/β-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/β-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/β-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/β-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/β-catenin signaling is important to the pathogenesis of gastrointestinal cancers.     

MicroRNA-140 attenuates myocardial ischemia-reperfusion injury through suppressing mitochondria-mediated apoptosis by targeting YES1

Myocardial ischemia-reperfusion (I/R) injury is thought to have its detrimental role in coronary heart disease (CHD), which is considered as the foremost cause of death all over the world. However, molecular mechanism in the progression of myocardial I/R injury is still unclear. The goal of this study was to investigate the expression and function of microRNA-140 (miR-140) in the process of myocardial I/R injury. The miR-140 expression level was analyzed in the myocardium with I/R injury and control myocardium using quantitative real-time polymerase chain reaction. Then the relation between the level of miR-140 and YES proto-oncogene 1 (YES1) was also investigated via luciferase reporter assay. Assessment of myocardial infarct size measurement of serum myocardial enzymes and electron microscopy analysis were used for analyzing the effect of miR-140 on myocardial I/R injury. We also used Western blot analysis to examine the expression levels of the mitochondrial fission–related proteins, Drp1 and Fis1. miR-140 is downregulated, and YES1 is upregulated after myocardial I/R injury. Overexpression of miR-140 could reduce the increase related to myocardial I/R injury in infarct size and myocardial enzymes, and it also could inhibit the expression of proteins related to mitochondrial morphology and myocardial I/R-induced mitochondrial apoptosis by targeting YES1. Taken together, these findings may provide a novel insight into the molecular mechanism of miR-140 and YES1 in the progression of myocardial I/R injury. MiR-140 might become a promising therapeutic target for treating myocardial I/R injury.     

Increased apoptosis of periprostatic adipose tissue in VDR null mice

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that controls mineral ion homeostatis and has potential tumor-suppressive functions for various cancer types, specifically prostate cancer. A VDR ablated transgenic animal model (VDDRII, vitamin D-dependent rickets type II) has been developed and the animals typically have various diseases including, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, and alopecia. This transgenic mouse system provides us with a model to decipher the influences of the VDR on prostatic growth and function. VDRs are abundant both in prostatic epithelial and stromal cells, and vitamin D signaling can be studied in this model. Although, there were no gross differences between the prostate tissue of the experimental and control groups, VDR null mice showed fat necrosis and individual cell apoptosis in the periprostatic adipose tissue. This indicates a possible role of VDR in the signaling pathways resulting the prostate. This may be particularly attractive for VDR targets for the inhibition of cancer progression using VD(3) and its analogs as potential chemo-preventive agents.