Aberrant expression of RAB1A in human tongue cancer

Abstract

This study was designed to identify specific gene expression changes in tongue squamous cell carcinomas (TSCCs) compared with normal tissues using in-house cDNA microarray that comprised of 2304 full-length cDNAs from a cDNA library prepared from normal oral tissues, primary oral cancers, and oral cancer cell lines. The genes identified by our microarray system were further analysed at the mRNA or protein expression level in a series of clinical samples by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis and imuunohositochemistry. The microarray analysis identified a total of 16 genes that were significantly upregulated in common among four TSCC specimens. Consistent with the results of the microarray, increased mRNA levels of selected genes with known molecular functions were found in the four TSCCs. Among genes identified, Rab1a, a member of the Ras oncogene family, was further analysed for its protein expression in 54 TSCCs and 13 premalignant lesions. We found a high prevalence of Rab1A-overexpression not only in TSCCs (98%) but also in premalignant lesions (93%). Thus, our results suggest that rapid characterisation of the target gene(s) for TSCCs can be accomplished using our in-house cDNA microarray analysis combined with the qRT-PCR and immunohistochemistry, and that the Rab1A is a potential biomarker of tongue carcinogenesis.

Figure 1

Validation of cDNA microarray data by real-time quantitative RT–PCR (qRT–PCR). (A) Nine genes with known molecular function were subjected to qRT–PCR in the mRNA from four TSCCs and four samples of the corresponding normal tissue used in the microarray analysis. A significant upregulation was evident in all the genes evaluated. (B) A significant higher expression of the Rab1a gene was detected in primary TSCCs (n=6) than that in the six corresponding normal tissues (P=0.0277, Wilcoxon signed-rank test). Relative expression ratio is defined as the expression levels of the gene to those of the internal reference gene, GAPDH. The assays were carried out in triplicate and means±standard deviations were plotted.

Figure 2

Immunohistochemical staining of Rab1A. (A) Normal tongue epithelium tissue shows weak expression of Rab1A protein. (B) Rab1A-positive case of TLP. The immunoreaction is slightly enhanced in the basal layer. (C) TSCC tissue shows strong cytoplasmic staining of the tumour cells. Original magnification, × 40. Bar=100 μm.

Figure 3

State of Rab1a protein expression in normal oral tissues (n=54), TLPs (n=13), and TSCCs (n=54). The Rab1A-IHC scores were calculated as follows: Rab1A-IHC=the mean percentage of positive tumour cells × staining intensity. Rab1A protein expressions in TLPs and TSCCs were significantly higher than in normal oral tissues (P<0.0001, Mann–Whitny’s U-test). There was also slight difference between TLPs and TSCCs (P=0.0268, Mann–Whitny’s U-test). Results are expressed as means±s.d.