Expression of p53, p63, podoplanin and Ki-67 in recurring versus non-recurring oral leukoplakia.

Oral leukoplakia (OL), a potentially malignant disorder, recurs in 40% of cases after surgical removal. Recurrence is a risk factor for malignant transformation. We aimed to examine the prognostic significance of four biomarkers related to cell proliferation: p53, p63, podoplanin (PDPN) and Ki-67 in predicting recurrence. Formalin-fixed-paraffin-embedded specimens from excised OL (n = 73, 33 recurrent; 40 non-recurrent) were collected in a prospective study. Immunohistochemistry was used to visualise expression of p53, p63, PDPN and Ki-67. Image analysis software was used for quantification of p53-, p63- and Ki-67-expressing cells, while PDPN was analysed visually. The expression of all four proteins were higher in recurrent compared with non-recurrent OL, only expression of p53 was statistically significant. In uni- and multivariable Cox regression analyses of individual markers, expression of p63 was significantly associated with higher recurrence risk (p = 0.047). OL with a combined high expression of both p53 and p63 had a significantly higher risk to recur [Log Rank, p = 0.036; multivariate Cox, HR: 2.48 (1.13-5.44; p = 0.024)]. Combination of p53 and p63 expression may be used as a prognostic biomarker for recurrence of OL.

Combined loss of expression of involucrin and cytokeratin 13 is associated with poor prognosis in squamous cell carcinoma of mobile tongue.

BACKGROUND: This study aimed to evaluate the prognostic significance of expression levels of involucrin (IVL), cytokeratin (CK)-10 and -13 at different intratumor sites (tumor center and invading area) of oral tongue squamous cell carcinoma (OTSCC). METHODS: IVL, CK13 and CK10 expression levels were examined in a multicenter cohort of 146 OTSCCs using immunohistochemistry. External mRNA datasets were used for expression analysis and/or to validate survival associations. RESULTS: External transcriptomic datasets showed downregulation of IVL and KRT13 in oral malignancies including OTSCC as compared to normal controls. The combined loss of IVL and CK13 expression at the invading core but not at the center core was significantly associated with poor differentiation and reduced 5-year overall survival. Multivariate Cox analysis confirmed the loss of CK13 and IVL expression to be an independent prognostic factor. Transcriptomic dataset corroborated immunohistochemistry results. CONCLUSIONS: Combined expression levlels of IVL and CK13 might be useful as prognostic biomarkers in OTSCC.

COVID-19 salivary signature: diagnostic and research opportunities.

The COVID-19 (caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) epidemic started in Wuhan (Hubei Province, China) in mid-December 2019 and quickly spread across the world as a pandemic. As a key to tracing the disease and to implement strategies aimed at breaking the chain of disease transmission, extensive testing for SARS-CoV-2 was suggested. Although nasopharyngeal/oropharyngeal swabs are the most commonly used biological samples for SARS-CoV-2 diagnosis, they have a number of limitations related to sample collection and healthcare personnel safety. In this context, saliva is emerging as a promising alternative to nasopharyngeal/oropharyngeal swabs for COVID-19 diagnosis and monitoring. Saliva collection, being a non-invasive approach with possibility for self-collection, circumvents to a great extent the limitations associated with the use of nasopharyngeal/oropharyngeal swabs. In addition, various salivary biomarkers including the salivary metabolomics offer a high promise to be useful for better understanding of COVID-19 and possibly in the identification of patients with various degrees of severity, including asymptomatic carriers. This review summarises the clinical and scientific basis for the potential use of saliva for COVID-19 diagnosis and disease monitoring. Additionally, we discuss saliva-based biomarkers and their potential clinical and research applications related to COVID-19.

Transcriptomic analysis of MicroRNA expression in enamel-producing cells.

There is little evidence for the involvement of microRNAs (miRNAs) in the regulation of circadian rhythms during enamel development. Few studies have used ameloblast-like cell line LS8 to study the circadian rhythm of gene activities related to enamel formation. However, the transcriptomic analysis of miRNA expression in LS8 cells has not been established yet. In this study, we analyze the oscillations of miRNAs in LS8 cells during one-day cycle of 24 h by next generation deep sequencing. After removal of low quality reads, contaminants, and ligation products, we obtained a high number of clean reads in all 12 samples from four different time points. The length distribution analysis indicated that 77.5% of clean reads were between 21 and 24 nucleotides (nt), of which 35.81% reads exhibited a length of 22 nt. In total, we identified 1471 miRNAs in LS8 cells throughout all four time-points. 1330 (90.41%) miRNAs were identified as known miRNA sequences, whereas 139 (9.59%) were unannotated and classified as novel miRNA sequences. The differential expression analysis showed that 191 known miRNAs exhibited significantly (P-value < 0.01) different levels of expression across three time-points investigated (T6, T12, and T18) compared to T0. Verification of sequencing data using qRT-PCR on six selected miRNAs suggested good correlation between the two methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of predicted target genes of differentially expressed miRNAs. The present study shows that miRNAs are highly expressed in LS8 cells and that a significant number of them oscillate during one-day cycle of 24 h. This is the first transcriptomic analysis of miRNAs in ameloblast-like cell line LS8 that can be potentially used to further characterize the epigenetic regulation of miRNAs during enamel formation.

Circadian clock and oral cancer.

The circadian clock is comprised of a master component situated in the hypothalamic suprachiasmatic nucleus and subordinate clock genes in almost every cell of the body. The circadian clock genes and their encoded proteins govern the organism to follow the natural signals of time, and adapt to external changes in the environment. The majority of physiological processes in mammals exhibit variable circadian rhythms, which are generated and coordinated by an oscillation in the expression of the clock genes. A number of studies have reported that alteration in the expression level of clock genes is correlated with several pathological conditions, including cancer. However, little is known about the role of clock genes in homeostasis of the oral epithelium and their disturbances in oral carcinogenesis. The present review summarizes the current state of knowledge of the implications of clock genes in oral cancer. It has been demonstrated that the development of oral squamous cell carcinoma undergoes circadian oscillation in relation to tumor volume and proliferation rate. The circadian clock gene period (PER)1 has been associated with oral cancer pathogenesis and it is suggested that changes in the expression of PER1 may exhibit an important role in the development, invasion, and metastasis of oral squamous cell carcinoma. However, its role remains elusive and there is a need for further research in order to understand the underlying mechanisms of the clock genes in oral cancer pathogenesis.

Circadian rhythms and gene expression during mouse molar tooth development.

OBJECTIVES: Incremental markings in dental enamel suggest that the circadian clock may influence the molecular underpinnings orchestrating enamel formation. The aim of this study was to investigate whether the genes and microRNAs (miRNAs) oscillate in a circadian pattern during tooth and enamel development. MATERIAL AND METHODS: Comparative gene and miRNA expression profiling of the first mandibular molar tooth germ isolated at different time-points during the light and night period was performed using microarrays and validated using real-time RT-PCR. Bioinformatic analysis was carried out using Ingenuity Pathway Analysis (IPA), and TargetScan software was used in order to identify computationally predicted miRNA-mRNA target relationships. RESULTS: In total, 439 genes and 32 miRNAs exhibited significantly different (p < 0.05) levels of expression in the light phase compared with the night phase tooth germs. Genes involved in enamel formation, i.e. Amelx, Ambn, Amtn, and Odam, oscillated in a circadian pattern. Furthermore, the circadian clock genes, in particular Clock and Bmal1, oscillated in mouse molar tooth germ during 24-h intervals. The expression of Clock and Bmal1 was inversely correlated with the expression of miR-182 and miR-141, respectively. CONCLUSIONS: MiRNAs, including miR-182 and miR-141, are involved in the control of peripheral circadian rhythms in the developing tooth by regulating the expression of genes coding for circadian transcription factors such as CLOCK and BMAL1. Regulation of circadian rhythms may be important for enamel phenotype, and the morphology of dental enamel may vary between individuals due to differences in circadian profiles.

Regulatory roles of microRNAs in human dental tissues.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that provide an efficient pathway for regulation of gene expression at a post-transcriptional level. Tooth development is regulated by a complex network of cell-cell signaling during all steps of organogenesis. Most of the congenital dental defects in humans are caused by mutations in genes involved in developmental regulatory networks. Whereas the developmental morphological stages of the tooth development already are thoroughly documented, the implicated genetic network is still under investigation. The involvement of miRNAs in the regulation of tooth genetic network was suggested for the first time in 2008. MiRNAs regulate tooth morphogenesis by fine-tuning the signaling networks. Unique groups of miRNAs are expressed in dental epithelium compared with mesenchyme, as well as in molars compared with incisors. The present review focuses on the current state of knowledge on the expression and function of miRNAs in human dental tissues, including teeth and the surrounding structures. Herein, we show that miRNAs exhibit specific roles in human dental tissues and are involved in gingival and periodontal disease, tooth movement and eruption, dental pulp physiology including repair and regeneration, differentiation of dental cells, and enamel mineralization. In light of similarities between the tooth development and other organs originating from the epithelium, further understanding of miRNAs` function in dental tissues may have wide biological relevance.

Prevalence of Epstein-Barr virus in oral squamous cell carcinoma, oral lichen planus, and normal oral mucosa.

OBJECTIVE: The Epstein-Barr virus (EBV) is associated with both malignant and benign diseases in the head and neck region. In several studies it has also been associated with oral squamous cell carcinoma (OSCC). Oral lichen planus (OLP) is a disease with unknown origin, and viral antigens have been proposed as etiologic agents. Smoking and alcohol habits are known risk factors for oral cancer development. In this study, the prevalence of EBV in OSCC and OLP was investigated, along with the effect of smoking, alcohol use, and age on EBV prevalence. STUDY DESIGN: We examined 29 patients with OSCC, 23 with OLP, and 67 with clinically healthy oral mucosa. For EBV DNA analysis, a nested polymerase chain reaction method was used. RESULTS: The overall EBV prevalence in patients with oral disease was 32.1%. Of the OSCC patients, 37.9% were EBV positive; and of the OLP patients, 26.1% were EBV positive. Both percentages were statistically significant compared with that of control patients (7.3%). The difference in EBV prevalence between the smoking control group and the nonsmoking control group was insignificant. Increased age did not enhance EBV prevalence. CONCLUSIONS: This investigation shows that EBV is present in oral diseases such as OSCC and OLP. Smoking, alcohol use, or age does not seem to be a risk factor for EBV infection. The etiologic role of EBV in OSCC and OLP needs to be examined in a prospective follow-up study.