Method optimization and gene expression analysis for interferon Kappa mRNA in HPV-associated cervical disease / by Correne A. Decarlo.
DeCarlo, Correne A.
MetadataShow full item record
Interferons (IFNs) are a common biological treatment option for many cancers as well as for human papillomavirus (HPV)-associated diseases, but current IFN treatments have not been optimized as disease progression subsists and treatment-limiting side effects still exist. IFN kappa (IFN-), a novel type I IFN, principally expressed in keratinocytes, may offer improvements in current IFN treatment regimes. The goal of the current study was to characterize the IFN-, IFN- and IFN- mRNA levels in normal, dysplastic and cervical carcinoma tissue, as well as microdissected cervical epithelium and stroma, to determine the effects of HPV on IFN- gene expression in relation to other common IFNs. Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring mRNA levels of low-expressing genes, such as IFNs, in human cervical samples are not described in the scientific literature. Thus, we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnoses were performed by one pathologist. Cervical keratinocytes and stroma were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. Human keratinocytes, cervical cancer-derived cell lines as well as patient-derived peripheral blood lymphocytes were used for supportive experiments. Here we report optimal methods for gene expression analysis of IFNs in cervical tissue as well as the comprehensive analysis of whole tissue and cell-specific IFN-y, -B and -K gene expression in HPV-associated cervical disease. IFN-K gene expression increased with disease progression, while IFN-B gene expression decreased with disease progression. IFN-y levels remained unchanged despite HPV infection. Furthermore, IFN-K expression in cervical stroma cells was induced upon HPV infection, implicating the involvement of cervical stroma cells in the HPV-associated increase in IFN-k expression. The described optimized methods could lead to improvements in immunological profiling and disease diagnosis, techniques without which IFN-k would remain undetectable in cervical samples. Further, characterization of the IFN immune profile in patients with different stages of HPV-associated cervical disease, with emphasis on the novel IFN-k, could help develop patient-tailored IFN treatment regimes that lead to the promotion of viral regression in women suffering from cervical disease.