MicroRNAs (miRNAs) are a class of ~22-nucleotide-long, non-coding RNAs widely expressed in eukaryotes and predominantly inhibit gene expression at the post-transcriptional level (1
). miRNAs control a wide range of biological processes, including, for instance, metabolism, organogenesis, development, cell growth, cell death and cell fate determination (5
). Furthermore, aberrant expression of miRNAs has been associated with human disease. For example, human miRNA expression signature can be used to distinguish between normal and cancerous lung tissues, with low let-7 expression and high miR-155 expression being correlated with poor prognosis for cancer patients (7
). How miRNAs contribute to tumorigenesis, however, is largely unknown.
Lung cancer is the most common cause of cancer death in the world, with an overall 5 years survival rate of only 14% upon diagnosis (12
). Approximately 90% of all lung cancers are directly attributable to smoking (14
). In the USA, despite considerable efforts over the last 40 years to reduce the prevalence of tobacco use, >45 million (22%) adults are still smokers (15
). Furthermore, former smokers are also at a higher risk of lung cancer than non-smokers. Development of alternate approaches to reduce lung cancer mortality, therefore, is a requisite public health issue, and our research explores two promising approaches, i.e. the identification of biomarkers for early detection and the development of effective chemopreventive agents.
Rodent models are invaluable tools for studying the initiation and progression of human disease, and we have been using these models to investigate the relationship between tobacco compounds and lung tumorigenesis (16
). To mimic tobacco carcinogenesis, rodents are fed with carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is a member of the nitrosamine family of compounds that, along with polycyclic aromatic hydrocarbons, are the most prevalent and potent carcinogens in tobacco products and smoke (17
). Inside a cell, NNK is first metabolically activated by α-hydroxylation reactions to produce metabolites such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, catalyzed by the cytochrome P450 (CYP) family of enzymes (20
). In the liver, the major P450 enzymes for NNK include CYP1A2, CYP2A6 and CYP3A4 in humans, whereas in the lung, rat CYP2A3, mouse CYP2A5 or human CYP2A13 is the most efficient catalyst. NNK metabolites then form alkylating adducts with DNA, leading to genetic mutations. NNK induces tumors in multiple organs and is a systemic lung carcinogen in laboratory animals, and it is a group 1 human carcinogen classified by the International Agency for Research on Cancer (21
Although dozens of miRNAs are differentially expressed in human lung cancer tissues compared with normal tissues (7
), how and at what stages they contribute to cancer development remains unclear. Lung cancer takes decades to develop in smokers, and once diagnosed, patients have a very low 5 year survival rate (12
). Thus, for prevention, diagnosis and treatment purposes, it is imperative to understand the early events accompanied with smoking, to which end animal models are essential. In this study, we examined alteration in miRNA expression at the early stages of lung cancer induction by obtaining miRNA expression profiles in the lungs of male F344 rats continuously fed with NNK for up to 20 weeks. Our goal was to generate a miRNA expression signature that could provide important, early diagnostic markers for lung cancers and to provide further insight into the etiology of NNK tumorigenesis.