Gene expression profiling of oral squamous cell carcinoma by differential display rt-PCR and identification of tumor biomarkers

Abstract

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Despite progress in therapeutic and surgical treatments, its survival period at 5 years is the lowest among major cancers, and remains unchanged in the last two decades. The growing epidemiological relevance of oral cancer emphasizes the need to better understand the molecular mechanisms underlying this disease and identify predictive tumor markers and therapeutic targets. To this end, we have used the DDRT-PCR analysis to profile the oral tumor transcriptome and identify differentially regulated genes that may be used as potential biomarkers and therapeutic targets. Our DDRT-PCR analysis identified 51 differentially expressed fragments, of which 25 were revalidated by reverse Northern analysis. Northern blot analysis further corroborated these findings for a few genes. In order to ascertain the utility of some of the identified genes as molecular markers and therapeutic targets, semi-quantitative RT-PCR analysis was carried out in a panel of matched oral normal and tumor samples, that confirmed GLTP, PCNA, RBM28, C17orf75 and DIAPH1 as significantly upregulated, whereas TNKS2, PAM and TUBB2C showed significant downregulation in tumor samples. Taken together, our DDRT-PCR analysis has revealed several genes, belonging to diverse cellular pathways, that have been associated with OSCC for the first time. Thus, these genes could be investigated as biomarkers and therapeutic targets for OSCC.

Keywords: Differential display RT-PCR; Gene expression; Molecular markers; OSCC; Oral cancer; Reverse northern; Squamous cell carcinoma.

Fig. 1

A representative differential display profile from a single patient using normal (N) and tumor (T) samples. T₁₁AC was used as the anchored primer. AP1, AP2 and AP3 were used as the arbitrary primers. The arrows point to bands showing differential expression pattern between the normal and tumor sample

Fig. 2

Clones identified as true differentials after second round of reverse Northern screening. Twelve clones were found to be upregulatedand and 13 were downregulated in tumor. ß-actin and GAPDH were used as equal loading controls. As expected, no change was observed in signal intensities for ß-actin and GAPDH spots probed with cDNA probes from normal or tumor tissues. ß-actin and GAPDH spots contain RT-PCR products. A >1.8 fold differential cut-off was used to designate the differential expression. Numbers in parentheses indicate the fold difference in expression in tumor in comparison to normal tissue

Fig. 3

Expression profiling of differentially expressed genes. a Northern blot hybridizations showing upregulation of PCNA, C17orf75 and ZKSCAN1, and downregulation of TUBB2C in the tumor sample. ß-actin was used as a control to equalize RNA loading. b Semi-quantitative RT-PCR analysis of genes identified as differentially expressed by DDRT-PCR analysis in 16 matched normal and tumor samples. Squares and triangles represent relative expression values for normal and tumor samples respectively, adjusted according to the internal control GAPDH. Each square or triangle corresponds to data from one sample. Horizontal lines represent mean values of mRNA expression across normal or tumor samples