MiRNA expression profiling and emergence of new prognostic signature for oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC), the most common type of head and neck cancers, is associated with high recurrence, metastasis, low long-term survival rates and poor treatment outcome. As deregulated miRNA expression plays a crucial role in malignant transformation and cancer progression, the present study is aimed at profiling the miRNA expression pattern in OSCC and developing a new miRNA prognostic signature for oral cancer. MiRNA expression profiling was performed using MiRNA microarray in 30 tumor and 18 normal samples. MiRNA signature obtained was validated with quantitative real time PCR (qRT-PCR) in 144 tumor and 36 normal samples. The potential targets, clinical implications and prognostic value of the miRNA signature were elucidated by various bioinformatics and statistical analyses. Microarray profiling identified a set of 105 miRNAs to be differentially expressed in OSCC, out of which a subset of 19 most dysregulated miRNAs were validated by qRT-PCR. In silico analysis revealed the signature miRNAs to be involved in various cancer associated pathways. Up-regulation of miR-196a, miR-21, miR-1237 and downregulation of miR-204, miR-144 was associated with poor prognosis of OSCC patients. The mir-196a/miR-204 expression ratio emerged as best predictor for disease recurrence and patient survival. Altogether, our study identified a miRNA signature for OSCC with prognostic significance.

Figure 1

(A) Supervised hierarchical clustering using signature miRNAs differentiating samples site-wise into distinct Tongue and Buccal mucosa clusters with cut off Fold Change > 1.5 and p value < 0.01. In the heatmap, samples are labelled with prefix ‘t’ for Tongue and ‘b’ for Buccal Mucosa (BM) and the suffix ‘C’ denotes Normals while the rest represents Tumor samples. (B) Heatmap showing the expression status of signature miRNAs in three external datasets compared to our profile; GSE28100 (USA, tongue), GSE31277 (Brazil, OSCC) and GSE4496 (USA, OSCC). Pink boxes represent upregulated miRNA, green boxes represent downregulated miRNA and yellow boxes indicates absence of miRNA expression.

Figure 2

Validated expression profiles of signature miRNAs compared to Clinico-Pathological characteristics of patient cohort. (A) Tumor recurrence (Green and orange bars represent non-recurrent and reccurent tumors respectively) (B) Disease Free Survival (green bars for good and blue bars for poor survival) and (C) Overall survival (Blue bars for average < 1 year, red for average 3 years and green for average 5 years).

Figure 3

(A) Expression ratio (normalized Ct) of miR-196a/miR-204 and its association with Disease-free and Overall survival. (B) ROC demonstrates the predictory power of miR-196a/miR-204 expression ratio.