Oral squamous cell carcinoma: microRNA expression profiling and integrative analyses for elucidation of tumourigenesis mechanism

Mayakannan Manikandan, Arungiri Kuha Deva Magendhra Rao, Ganesan Arunkumar, Meenakshisundaram Manickavasagam, Kottayasamy Seenivasagam Rajkumar, Ramamurthy Rajaraman, Arasambattu Kannan Munirajan

The advantages and utility of microRNAs (miRNAs) as diagnostic and prognostic cancer markers is at the vanguard in recent years. In this study, we attempted to identify and validate the differential expression of miRNAs in oral squamous cell carcinoma (OSCC), to correlate their expression with the clinico-pathological profile of tumours and to identify the signaling pathways through which the aberrantly expressed miRNAs effect tumourigenesis. miRCURY LNA™ array with probes specific to 1168 miRNAs and TaqMan assays specific for 10 miRNAs was employed to evaluate and validate miRNA expression in a discovery cohort (n = 29) and validation cohort (n = 61) of primary OSCC tissue specimens, respectively. A computational pipeline with sequential integration of data from miRTarBase, CytoScape, UniProtKB and DIANA-miRPath was utilized to map the target genes of deregulated miRNAs and associated molecular pathways. Microarray profiling identified 46 miRNAs that were differentially expressed in OSCC. Unsupervised clustering demonstrated a high degree of molecular heterogeneity across the tumour samples as the clusters did not represent any of their clinico-pathological characteristics. The differential expression of 10 miRNAs were validated by RT-qPCR (let-7a, let-7d, let-7f and miR-16 were downregulated while miR-29b, miR-142-3p, miR-144, miR-203, and miR-223 were upregulated in OSCC; the expression of miR-1275 was variable in tumours, with high levels associated to regional lymph node invasion; additionally, miR-223 exhibited an association with advanced tumour stage/size). In silico analyses of the experimentally confirmed target genes of miRNAs revamp the relationship of upregulated miRNAs with tumour suppressor genes and of downregulated miRNAs with oncogenes. Further, the differentially expressed miRNAs may play a role by simultaneously activating genes of PI3K/Akt signaling on one hand and by repressing genes of p53 signaling pathway on the other. The identified differentially expressed miRNAs and signaling pathways deregulated in OSCC have implications for the development of novel therapeutic strategies. To the best of our knowledge, this is the first report to show the association of miR-1275 with nodal invasion and the upregulation of miR-144 in OSCC.