A potential association between mutations in the iNOS cDNA 3′ stretch and oral squamous cell carcinoma - A preliminary study

Oral cancers are linked with tobacco, areca nut and alcohol addictive proclivities. The oral squamous cell carcinoma (OSCC) comprises more than 90% cases of oral cancers. The Homo sapiens inducible nitric oxide synthase (iNOS) gene plays crucial role in OSCC pathologies. The given study analyzed the nucleotide mutations existent within two targets of interest namely iNOS cDNA 5′ stretch and iNOS cDNA 3′ stretch, using cDNA pool of cancer and pre-cancer oral biopsies employing Q PCR-Tm calling approach. The iNOS cDNA 5′ stretch encompassed exons 4 and 5 and iNOS cDNA 3′ stretch embodied exons 18, 19, and 20. Total RNA was isolated from oral biopsies: cancer biopsies (OSCC lesions - adjacent normal tissue (ANT) from fifteen OSCC cases), pre-cancer biopsy (ANT of chronic inflamed lesion) and normal oral mucosa (control). The total RNA samples were quantified and PCR based assurance was accomplished for genomic DNA absence. The PCR based integrity assessment was performed using synthesized cDNA samples. The Q PCR-Tm calling was performed using specific primers for normalizer gene (GAPDH) and the targets of interest. The amplicons for normalizer gene and iNOS cDNA 5′ stretch respectively showed Tm values of ~80 °C and ~81 °C in both control and diseased cDNA pools. On the contrary, the iNOS cDNA 3′ stretch showed amplicons with multiple Tm values in diseased (pre-cancer and cancer lesions) cDNA pools, varying significantly from the control (~85 °C). The results indicate existence of highly mutated iNOS cDNA 3′ stretches in the cDNA pool of oral cancer and pre-cancer lesions and adjoining visibly normal tissues, thereby raising a need for rigorous experimental efforts for unraveling the functional association(s) between the mutations in iNOS cDNA 3′ stretch and oral precancer-cancer pathophysiologies.