A Role for MicroRNA-155 Expression in Microenvironment Associated to HPV-Induced Carcinogenesis in K14-HPV16 Transgenic Mice

January A Role for MicroRNA-155 Expression in Microenvironment Associated to HPV- Induced Carcinogenesis in K14-HPV16 Transgenic Mice Isabel Paiva 0 1 2 Rui M. Gil da Costa 0 1 2 Joana Ribeiro 0 1 2 Hugo Sousa 0 1 2 Margarida Bastos 0 1 2 Ana Faustino Carlos Rocha 0 1 2 Paula A Oliveira 0 1 2 Rui Medeiros 0 1 2 0 1 Molecular Oncology and Viral Pathology Group, CI-IPOP, Portuguese Institute of Oncology of Porto , Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal, 2 ICBAS , Abel Salazar Institute for the Biomedical Sciences, University of Porto , Rua de Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal, 3 LEPABE , Faculty of Engineering, University of Porto , Rua Dr. Roberto Frias s/n, 4200-465, Porto , Portugal , 4 Experimental Pathology and Therapeutics Group, CI-IPOP, Portuguese Institute of Oncology , Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto , Portugal , 5 Virology Service, Portuguese Institute of Oncology of Porto , Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto , Portugal , 6 Veterinary Sciences Department, University of Tras-os-Montes and Alto Douro, UTAD, Quinta de Prados , 5001-801, Vila Real , Portugal , 7 Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tras-os-Montes and Alto Douro, UTAD, Quinta de Prados , 5001-911, Vila Real, Portugal, 8 CEBIMED , Faculty of Health Sciences of Fernando Pessoa University , Porto , Portugal , 9 Portuguese League Against Cancer (Liga Portuguesa Contra o Cancro-Nucleo Regional do Norte) , Estrada Interior da Circunvalacao, no6657, 4200-177 Porto , Portugal 1 Data Availability Statement: All relevant data are in the Supporting Information file (Supporting Information(Table S1) 2 Academic Editor: George Calin, University of Texas, MD Anderson Cancer Center , UNITED STATES Human Papillomavirus cause a number of diseases most notably cervical cancer. K14HPV16 transgenic mice expressing the HPV16 early genes in squamous epithelial cells provide a suitable experimental model for studying these diseases. MicroRNAs are small non-coding RNAs that play an important role in regulating gene expression and have been suggested to play an important role in cancer development. The role of miR-155 in cancer remains controversial and there is limited evidence linking this miRNA to HPV- associated diseases. We hypothesized that miR-155 expression modulates each tissue's susceptibility to develop HPV-associated carcinogenesis. In this study, we analyzed miR-155 expression in ear and chest skin samples from 22-26 weeks old, female K14-HPV16 transgenic (HPV16+/-) and wild-type (HPV-/-) mice. Among wild-type mice the expression of miR-155 was lower in ear skin compared with chest skin (p = 0.028). In transgenic animals, in situ perplasia. Furthermore, in hyperplastic chest skin samples, miR-155 expression was lower than in normal chest skin (p = 0,026). These results suggest that miR-155 expression may modulate the microenvironmental susceptibility to cancer development and that high miR155 levels may be protective against the carcinogenesis induced by HPV16. - Funding: This study was supported by Liga Portuguesa Contra o Cancro. Rui M. Gil da Costa is supported by grant SFRH/BPD/85462/2012 from the Portuguese Foundation for Science and Technology, financed by the Portuguese Government and the Social European Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Human papillomaviruses (HPVs) are the most common sexually transmitted agents [1]. Highrisk human papillomavirus, such as HPV16 and HPV18, are the causative agents of virtually all cases of cervical cancer and a significant proportion of other anogenital cancers, as well as some head and neck cancers [24]. The K14-HPV16 transgenic mouse model is particularly useful to study the development of HPV-associated squamous cells carcinomas. In this model, the expression of HPV16 early region genes (E2-E8) is driven by the cytokeratin 14 (K14) promoter/enhancer, specifically targeting epithelial basal cells [5]. Basal cells are mitotically active and thus may develop further mutations in response to a proliferative stimulus, and the expression of K14 has been shown to persist in well-differentiated squamous carcinomas [6]. The expression of the HPV oncogenes E6 and E7 induces epithelial carcinogenesis through multiple premalignant stages [7]. Accordingly, the K14-HPV16 transgenic mice develop epidermal hyperplasia that progresses to dysplasia and in situ carcinoma (CIS) lesions and, ultimately, to invasive cancer. This animal model simulates the multistep carcinogenesis process and thus facilitates the study of epigenetic and the genetic factors that coordinate malignant conversion and regulate neoplastic progression. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression promoting inhibition of messenger RNA (mRNA) translation or its degradation [8]. In normal cells, miRNAs control several processes including proliferation, differentiation and apoptosis. These molecules are also described as key regulators in many diseases including neurological disorders, cardiovascular diseases, viral infections and cancer [9]. During carcinogenesis, some miRNAs are lost whereas others are upregulated, and in fact, previous data indicates that miRNAs may be important to distinguish subtypes of cancers, where the histological diagnosis is complex and difficult [10]. MicroRNA-155 (miR-155) plays a role in many of the above oncogenic processes. This microRNA is overexpressed in many types of cancer cells and accumulating evidence shows that miR-155 is an oncogenic microRNA. However, recent studies claim that miR-155 may display anti-oncogenic properties or promote an adequate immunological response to cancer [11,12]. MiR-155 has emerged as an essential regulator of cellular physiology, particularly important in the mammalian immune system [1315]. Thus, a possible link between miR-155 and inflammation in cancer has been reported [16]. Moreover, miR-155 transgenic mice develop B-cell lymphoma [15], and miR-155-knock-out mice exhibit impaired immune function [17]. In HPV-associated cancers, the interplay between miR-155 and HPV genes remains elusive and poorly understood. The tumor microenvironment associated to miRNAs plays an increasingly appreciated role in cancer [18], but the microenvironment of normal tissues and its role in tumorigenesis remains poorly studied. In this study, we aimed to evaluate the expression of miR-155 in skin samples with or without the presence of integrated HPV DNA and with different HPV-associated lesions. For this purpose, we have used K14-HPV16 transgenic mice, [19], to analyze miR-155 expression in ear and chest skin samples, evaluating its correlation with tissue microenvironment and HPVinduced carcinogenesis. Materials and Methods Transgenic mice Generation of K14-HPV mice has been p (...truncated)