Method: Expression of miR-196a in normal (NOK), immortalised normal (iNOK), oral pre-malignant (OPM) keratinocytes and HNSCC-derived cell lines was assessed by quantitative-RTPCR (qRTPCR). Laser capture microdissection (LCM) followed by qRTPCR was used to measure miR-196a expression in FFPE tissue samples. Anti-miR-196a was transfected into OPM and HNSCC cells followed by functional assays for adhesion to fibronectin, proliferation (MTS assay), invasion and migration (modified transwell). Microarray analysis of anti-miR-196a and pre-miR-196a transfected cells was conducted using Agilent Sureprint G3 array and gene expression changes seen were validated by qRTPCR. The 3’UTR of novel targets were cloned into pMIR REPORT and dual reporter luciferase assay was performed as per manufacturer’s protocol.
Result: 600-4000 fold up-regulation of miR-196a was observed in OPM and HNSCC cells compared to NOK’s. FFPE cancer tissue samples showed significant up-regulation compared to normal tissue for miR-196a (p<0.05). Transfection of anti-miR-196a resulted in reduced adhesion, invasion and migration (p<0.001), but no change in proliferation of HNSCC cells. Microarray analysis and qRTPCR validation showed HOXC8, MAMDC2 and RFC3 to be putative targets of miR-196a. Dual reporter luciferase assay confirmed MAMDC2 to be direct target of miR-196a (p<0.001).
Conclusion: Our data shows that miR-196a is over-expressed and promotes functional effects in HNSCC, thus miR-196a could be used as diagnostic marker in HNSCC. We have identified a novel miR-196a target gene, MAMDC2, which requires further investigation of its function.