Gene Expression Analysis of Human Papillomavirus-Associated Colorectal Carcinoma

Abstract

Purpose: Human papillomavirus (HPV) antigens had been found in colorectal cancer (CRC) tissue, but little evidence demonstrates the association of HPV with oncogene mutations in CRC. We aim to elucidate the mutated genes that link HPV infection and CRC carcinogenesis.

Methods: Cancerous and adjacent noncancerous tissues were obtained from CRC patients. HPV antigen was measured by using the immunohistochemical (IHC) technique. The differentially expressed genes (DEGs) in HPV-positive and HPV-negative tumor tissues were measured by using TaqMan Array Plates. The target genes were validated with the qPCR method.

Results: 15 (31.9%) cases of CRC patients were observed to be HPV positive, in which HPV antigen was expressed in most tumor tissues rather than in adjacent noncancerous tissues. With TaqMan Array Plates analyses, we found that 39 differentially expressed genes (DEGs) were upregulated, while 17 DEGs were downregulated in HPV-positive CRC tissues compared with HPV-negative tissues. Four DEGs (MMP-7, MYC, WNT-5A, and AXIN2) were upregulated in tumor vs. normal tissues, or adenoma vs. normal tissue in TCGA, which was overlapped with our data. In the confirmation test, MMP-7, MYC, WNT-5A, and AXIN2 were upregulated in cancerous tissue compared with adjacent noncancerous tissue. MYC, WNT-5A, and AXIN2 were shown to be upregulated in HPV-positive CRC tissues when compared to HPV-negative tissues.

Conclusion: HPV-encoding genome may integrate into the tumor genomes that involved in multiple signaling pathways. Further genomic and proteomic investigation is necessary for obtaining a more comprehensive knowledge of signaling pathways associated with the CRC carcinogenesis.

Figure 1

Distribution of HPV viral antigen in CRC tumor tissue and adjacent nonneoplastic tissue by immunohistochemistry staining. (a) HPV-negative expression in the neoplastic tissue of CRC patients with TNM stage 1. (b) HPV-positive squamous cell in cervical carcinoma was the positive control. (c, d) Patient 1 (TNM stage 3), with sigmoid colon cancer by which the pathological type is ulcerative moderately differentiated adenocarcinoma, and tumor cells infiltrate to the serous layer and adipose tissue. HPV staining is positive in tumor tissue (d) but negative in adjacent nonneoplastic tissue (c). (e, f) Patient 2 (TNM stage 3), with rectal cancer by which the pathological type is ulcerative moderately differentiated adenocarcinoma, and tumor cells infiltrate to the outer membrane; HPV staining is positive in tumor tissue (f) but negative in adjacent nonneoplastic tissue (e).

Figure 2

The heat map of 23 differentially expressed genes (DEGs) in HPV-positive and HPV-negative CRC tissues. RQ7 and RQ9 were tumor tissues with HPV negative. RQ4 and RQ12 were tumor tissues with HPV positive. The gene expression data were measured with microarray methods. Relative expression values (2-ΔCt) of DEGs from HPV-positive/negative sample were calculated and generated the heat map. DEG expression was shown with different colors. From green to red represent downregulated to upregulated genes.

Figure 3

Figure 4

Expression of AXIN2, MYC, MMP7, and WNT-5A in CRC tissue and adjacent noncancerous tissue.

Figure 5

Different expression levels of AXIN2, MYC, MMP7, and WNT-5A between HPV-positive and HPV-negative tumor tissues.

Figure 6

Principal component analysis (PCA) of gene expression data for HPV-positive and HPV-negative CRC tissues. RQ7 and RQ9 were tumor tissues with HPV negative. RQ4 and RQ12 were tumor tissues with HPV positive. 4 CRC samples (2 from viral-positive group and 2 from viral-negative group) were significantly separated into two areas.

Figure 7

Two-way hierarchical clustering analysis of the data downloaded from TCGA. Group C for CRC tissues and Group N for normal tissues. The results showed that 92 genes were significantly separated into two areas: tumor tissue vs. normal tissue.