Multiple Signatures of the JC Polyomavirus in Paired Normal and Altered Colorectal Mucosa Indicate a Link with Human Colorectal Cancer, but Not with Cancer Progression

Abstract

The JC polyomavirus (JCV) has been repeatedly but discordantly detected in healthy colonic mucosa, adenomatous polyps, and colorectal cancer (CRC), and proposed to contribute to oncogenesis. The controversies may derive from differences in JCV targets, patient's cohorts, and methods. Studies of simultaneous detection, quantification, and characterization of JCV presence/expression in paired samples of normal/altered tissues of the same patient are lacking. Therefore, we simultaneously quantified JCV presence (DNA) and expression (mRNA and protein) of T-antigen (T-Ag), Viral Protein 1 (Vp1), and miR-J1-5p in paired normal/altered tissues of CRC or polyps, and from controls. JCV signatures were found in most samples. They increased in patients, but were higher in normal mucosa than in corresponding polyp or CRC lesions. JCV non-coding control region (NCCR) DNA rearrangements increased in CRC patients, also in normal mucosa, thus before the onset of the lesion. A new ∆98bp NCCR DNA rearrangement was detected. T-Ag levels were higher in normal mucosa than in adenoma and adenocarcinoma lesions, but decreased to levels of controls in established CRC lesions. In CRC, miR-J1-5p expression decreased with CRC progression. Vp1 expression was not detected. The data indicate a JCV link with the disease, but possible JCV contributes to oncogenesis should occur at pre-polyp stages.

Keywords: JC polyomavirus; NCCR non-coding control region; T-antigen; adenomatous polyps; colorectal cancer; oncogenesis.

Figure 1

Levels of T-antigen (T-Ag) DNA in NTC, polyps, and CRC tumor and normal surrounding tissues. (A) Mean values of JCV T-Ag DNA copies in NTC, polyps, and CRC tumor (T) and normal (N) surrounding tissues; (B) individual values of JCV T-Ag DNA copies of paired tumor (T) and normal (N) surrounding tissues in CRC cases. Data are expressed according to the 2^−∆Ct method [39]. The statistical significance was determined by the two-tailed Student’s t-test.

Figure 2

Presence and characterization of the JC polyomavirus (JCV) NCCR DNA in NTC, polyps, and CRC pairs. (A) Migration on agarose gels of representative NCCR amplicons, obtained by nested PCR, as specified in the Methods section. M: marker; +: positive control; NTC: non tumor control; P: polyp; N: normal adjacent mucosa; T: tumor mucosa. (B) NCCR multiple alignment of sample sequences to JCV strains from the NCBI database. At the top, a schematic representation of the PML-type NCCR organization is reported. (C) Distribution of the Mad-1 prototype and Δ98 rearranged NCCR forms in NTC, polyps, and CRC, expressed as percentage of cases. See text for details.

Figure 3

T-Ag expression in NTC, polyps, and CRC pairs. (A) Levels of T-Ag transcripts in paired adenomatous polyps and CRC cases. (B) Levels of T-Ag protein in CRC for each patient. The gray lines indicate the two samples with higher levels in the lesion than in normal tissue. (C) Western blotting of representative samples from non-tumor controls (NTC), polyps (P), and CRC normal (N) and tumor (T) tissues. (D) Arbitrary quantification of the intensity of relevant bands in western blotting for each sample of NTC, polyps, and CRC. The levels of T-Ag transcripts were expressed according to the 2^−∆Ct method [39]. The statistical significance was determined by the two-tailed Student’s t-test in (A) and (D), and by the Wilcoxon signed rank test for paired samples in (B).

Figure 4

Levels of miR-J1-5p microRNA (miRNA) in CRC pairs. (A) miR-J1-5p values, obtained by a quantitative stem-loop RT-PCR miRNA assay, and expressed as CRC tumor/normal ratio. The values are sorted in ascending order. (B) Relationship among JCV miR-J1-5p miRNA and T-Ag expression; the relative miRNA expression in tumor and normal tissues (T/N ratio) is plotted against the corresponding T-Ag relative expression, expressed as T/N ratio. (C) miR-J1-5p CRC tumor/normal ratios stratified according to CRC TNM staging. The statistical significance was determined by two-tailed Student’s t-test.