A novel 3-miRNA network regulates tumour progression in oral squamous cell carcinoma.

BACKGROUND: Late diagnosis is one of the major confounders in oral squamous cell carcinoma (OSCC). Despite recent advances in molecular diagnostics, no disease-specific biomarkers are clinically available for early risk prediction of OSCC. Therefore, it is important to identify robust biomarkers that are detectable using non-invasive liquid biopsy techniques to facilitate the early diagnosis of oral cancer. This study identified potential salivary exosome-derived miRNA biomarkers and crucial miRNA-mRNA networks/underlying mechanisms responsible for OSCC progression. METHODS: Small RNASeq (n = 23) was performed in order to identify potential miRNA biomarkers in both tissue and salivary exosomes derived from OSCC patients. Further, integrated analysis of The Cancer Genome Atlas (TCGA) datasets (n = 114), qPCR validation on larger patient cohorts (n = 70) and statistical analysis with various clinicopathological parameters was conducted to assess the effectiveness of the identified miRNA signature. miRNA-mRNA networks and pathway analysis was conducted by integrating the transcriptome sequencing and TCGA data. The OECM-1 cell line was transfected with the identified miRNA signature in order to observe its effect on various functional mechanisms such as cell proliferation, cell cycle, apoptosis, invasive as well as migratory potential and the downstream signaling pathways regulated by these miRNA-mRNA networks. RESULTS: Small RNASeq and TCGA data identified 12 differentially expressed miRNAs in OSCC patients compared to controls. On validating these findings in a larger cohort of patients, miR-140-5p, miR-143-5p, and miR-145-5p were found to be significantly downregulated. This 3-miRNA signature demonstrated higher efficacy in predicting disease progression and clinically correlated with poor prognosis (p < 0.05). Transcriptome, TCGA, and miRNA-mRNA network analysis identified HIF1a, CDH1, CD44, EGFR, and CCND1 as hub genes regulated by the miRNA signature. Further, transfection-mediated upregulation of the 3-miRNA signature significantly decreased cell proliferation, induced apoptosis, resulted in G2/M phase cell cycle arrest and reduced the invasive and migratory potential by reversing the EMT process in the OECM-1 cell line. CONCLUSIONS: Thus, this study identifies a 3-miRNA signature that can be utilized as a potential biomarker for predicting disease progression of OSCC and uncovers the underlying mechanisms responsible for converting a normal epithelial cell into a malignant phenotype.

Salivary Exosomal miRNA-1307-5p Predicts Disease Aggressiveness and Poor Prognosis in Oral Squamous Cell Carcinoma Patients.

Background: Salivary exosomal miRNAs as biomarkers facilitate repeated sampling, real-time disease monitoring and assessment of therapeutic response. This study identifies a single salivary exosomal miRNA prognosticator that will aid in improved patient outcome using a liquid biopsy approach. Method: Small RNA and transcriptome sequencing profiles of tumour tissues (n = 12) and salivary exosomes (n = 8) from oral cancer patients were compared to their non-cancerous counterparts. We validated these results using The Cancer Genome Atlas database and performing Real-time PCR on a large patient cohort (n = 19 tissue samples; n = 12 salivary exosomes). Potential target genes and the miRNA-mRNA networks and enriched biological pathways regulated by this microRNA were identified using computational tools. Results: Salivary exosomes (size: 30-50 nm) demonstrated a strong expression of CD47 and detectable expression of tetraspanins CD63, CD81 and CD9 by flow cytometry. miR-1307-5p was exclusively overexpressed in tissues and salivary exosomes of oral cancer patients compared to their non-cancerous counterparts. Enhanced expression of miR-1307-5p clinically correlated with poor patient survival, disease progression, aggressiveness and chemo-resistance. Transcriptome analysis suggested that miRNA-1307-5p could promote oral cancer progression by suppressing THOP1, EHF, RNF4, GET4 and RNF114. Conclusions: Salivary exosomal miRNA-1307-5p is a potential prognosticator for predicting poor survival and poor patient outcome in oral cancers.

Saliva Based Liquid Biopsies in Head and Neck Cancer: How Far Are We From the Clinic?

Head and neck cancer (HNC) remains to be a major cause of mortality worldwide because of confounding factors such as late-stage tumor diagnosis, loco-regional aggressiveness and distant metastasis. The current standardized diagnostic regime for HNC is tissue biopsy which fails to determine the thorough tumor dynamics. Therefore, due to the ease of collection, recent studies have focused on the utility of saliva based liquid biopsy approach for serial sampling, early diagnosis, prognosis, longitudinal monitoring of disease progression and treatment response in HNC patients. Saliva collection is convenient, non-invasive, and pain-free and offers repetitive sampling along with real time monitoring of the disease. Moreover, the detection, isolation and analysis of tumor-derived components such as Circulating Tumor Nucleic Acids (CTNAs), Extracellular Vesicles (EVs), Circulating Tumor Cells (CTCs) and metabolites from saliva can be used for genomic and proteomic examination of HNC patients. Although, these circulatory biomarkers have a wide range of applications in clinical settings, no validated data has yet been established for their usage in clinical practice for HNC. Improvements in isolation and detection technologies and next-generation sequencing analysis have resolved many technological hurdles, allowing a wide range of saliva based liquid biopsy application in clinical backgrounds. Thus, in this review, we discussed the rationality of saliva as plausible biofluid and clinical sample for diagnosis, prognosis and therapeutics of HNC. We have described the molecular components of saliva that could mirror the disease status, recent outcomes of salivaomics associated with HNC and current technologies which have the potential to improve the clinical value of saliva in HNC.