Detection of EBV infection and gene expression in oral cancer from patients in Taiwan by microarray analysis

Abstract

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.

Figure 1

Production of EBV-chip for hybridizing with cDNA samples derived from OSCC biopsies. (a) 16 control and 71 EBV DNA fragments were organized into an 8-row by 12-column format with the size of 4.2 × 2.4 mm, as specified in the previous report [29]. The controls are located at the upper left corner included in the square box, where it contains 12 DNA fragments for 1.2 and 1.1 kbp Pst I-digested-lambda bacteriophage DNA, plant APS1, ASA1, GA4, HAT4, HAT22, LhcI, RbcL, and Rca genes, and human GAPDH and β-actin genes. In addition, there were four dye spots that served as the negative controls. Outside the control region, 71 EBV DNA spots, with the gene names, covering the entire EBV genome were arranged in a sequential order on EBV-chip, except that spot R was interrupted between Spots 6 and 27. Spots R and 90r contained EBV W- (internal) and terminal-repeated, respectively, sequences. The arrangement of viral DNA on EBV-chip could facilitate identification of expressed genes shown in hybridization images. (b) Total RNA samples were isolated from U937 cell line, EBV-producing B95-8 cell line, and P3HR1 cell line treated without (−) or with TPA and SB to induce the EBV lytic cycle (+), followed by EBV-chip hybridizations. One of each of the representative reproducible images of these hybridization experiments is shown in here, with the control regions boxed. Since U937 cells were EBV-free, no specific signal on EBV-chip spot was detected (the light color in 90r was the nonspecific hybridization signal, as discussed in the Results section). The RNA samples derived from B95-8 and P3HR1 (+) cells contained EBV transcripts, and therefore lots of, if not all, spots showed dark color, whereas a limited number of spots yielded signals when using EBV-latent P3HR1 (−) cDNA in hybridizations. (c) Cases numbers 41 and 54 were among those 10 EBV-negative OSCC tissues, and basically EBV-chip hybridizations of their cDNA samples revealed no expression signal. (d) Two EBV-positive tissues, numbers 9 and 28, contained substantial numbers of EBV transcripts that resulted in many dark spots in EBV-chips. The intensities of the spots were subsequently quantified and subjected to comparative analysis, as shown in Figure 2.

Figure 2

Hierarchical presentation of EBV gene expression patterns in forty-seven OSCC specimens. The EBV gene expression patterns in the OSCC tissues were presented by colored codes, as pink, red, blue, green, and yellow squares represented log numbers in the respective ranges of 5.9–5.0, 4.9–4.0, 3.9–3.0, 2.9–2.0, 1.9–1.0, and < 0, with larger numbers denoting greater expression levels. Among the 47 tissue specimens, the total numbers of samples that expressed the particular viral genes were counted and listed on the left (No. exp. in tissues).

Figure 3

Quantitative real-time RT-PCR of the CD45, EBNA1, and EBNA2 transcripts in tumor samples. Due to the limited number of available frozen tissues, seven OSCC samples numbered 38, 41, 2, 47, 13, 14, and 21 (Case numbers are as those listed in Table 1), with the former two categorized as EBV-negative and others as positive, are used in this quantitative real-time PCR experiment. The portions in the frozen tissues applying to the experiments were all at positions adjacent to the respective tissue specimens used in the prior EBV-chip hybridizations. The results reveal that, after normalizing with the expression levels for the β-actin gene, the expressions of the gene for the pan-WBC surface marker CD45 in both the cell lines U937 and B95-8 are clearly detected, whereas they are low in all cancer samples, suggesting that EBV gene expression, if any, present in infiltrated blood cells does not contribute to the signals detected with EBV-chip. Tumor sample numbers 38 and 41 are EBV-negative (Table 3), samples 2 and 47 only express the great amounts of the EBNA2 gene transcript, and the rest of cases numbered 13, 14, and 21 have the strong expression signals for both the EBNA1 and EBNA2 genes. In conclusion, the data from the quantitative real-time RT-PCR analysis are in agreement with those shown in Figure 2. In here, two identical quantitative real-time PCR have been performed and very similar results are obtained. □: CD45; : EBNA1; ■: EBNA2.