Identification of Differentially Expressed Genes in HPV AssociatedCancers Using Gene Expression, Tissue,and MicroRNA Microarrays

Infections with high-risk human papillomaviruses (HPVs) have been implicated in the pathogenesis of cervical carcinoma and a subset of squamous cell carcinoma of the head and neck (SCCHN). In this study, we compared the cellular gene expression profiles of HPV16-positive and HPV-negative oropharyngeal carcinomas with those of the normal oral epithelium. Using a high-density oligonucleotide microarray containing 22,215 human transcripts, we showed that 397 and 162 genes were differentially expressed in HPV16-positive and HPV-negative SCCHN, respectively, compared to the normal oral epithelium. Our studies also identified 59 differentially expressed genes in HPV16-positive SCCHN as compared to both HPV-negative SCCHN and normal oropharyngeal tissues. Such up-regulated genes included those involved in nuclear structure and meiosis (SYCP2), DNA repair (RFC5), and transcription regulation (ZNF238). Genes involved in proteolysis (KLK8) and signal transduction (CRABP2) were found to be down-regulated in HPV-positive SCCHN. Our results reveal specific gene expression patterns in HPV16-positive oropharyngeal squamous carcinomas and suggest that HPV infection could play an important etiologic role in these tumors. In another study using the same high-density microarray platform, we have analyzed the cellular gene expression profiles of five HPV-16 and two HPV-18 positive cervical cell lines, one HPV-negative cervical carcinoma cell line, and normal cervical tissue. Our results showed that 877 and 536 genes were differentially expressed in the HPV-positive cell lines compared to the normal cervix tissue and the HPV-negative cervical carcinoma cell line C-33A, respectively. We also found that a total of 57 genes were differentially expressed in the HPV-positive cell lines as compared to both the normal cervix and C-33A. Differentially expressed genes including those involved in cell proliferation such as the L-type amino acid transporter 1 (LAT1, also known as SLC7A5) and gene expression regulation like nucleosome assembly protein 1-like 3 (NAP1L3) were found to be affected for the first time in cervical cell lines. In situ hybridization of LAT1 and NAP1L3 mRNA performed using tissue-arrays (containing ~50 different cervical tumor samples per slide) showed that these genes are also affected in their expression in tumor tissues. These results could lead to the identification of new cellular pathways affected by the presence of HPV in cervical cells. We have also carried out studies to determine whether the expression of human microRNAs (miRNAs; small non-coding RNAs that have the ability to regulate gene expression) are affected by the presence of HPV DNA. For this purpose, we analyzed the expression of miRNAs in HPV-16 positive cervical cell lines and tissues. Twenty-seven miRNAs were differentially expressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix, while, only 6 miRNAs were differentially expressed in a cell line containing episomal HPV-16 DNA compared to the normal cervix. Furthermore, 10 miRNAs were affected in their expression in cell lines containing integrated HPV-16 DNA compared to C-33A. Interestingly, microRNA-218 (miR-218) was specifically underexpressed in cell lines, cervical lesions and cancer tissues containing integrated HPV-16 DNA as compared to both the HPV-negative cell line C-33A and the normal cervix. Expression of the HPV-16 E6 oncogene in transfected cells reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16 positive cell line increased miR-218 expression. We also showed that miR-218 expression parallels that of the tumor suppressor gene SLIT2 whose intron encodes miR-218. Furthermore, exogenous expression of miR-218 in HPV-16 positive cell lines decreased expression of the epithelial-specific gene LAMB3 which is involved in cell migration and tumorigenicity. These findings demonstrate specific regulation of cellular miRNAs in the presence of an HPV oncogene and may contribute to a better understanding of molecular mechanisms involved in cervical carcinogenesis