Taken collectively, the body of literature describing our understanding of the effects of cigarette smoke on ncRNA expression and function is very much in its infancy. Studies to date have been relatively small in scope, have considered a relatively limited number of target tissues (i.e., placenta, lung, liver, and airway epithelial cells), and examined various types of cigarette smoke exposure (e.g., primary, passive/secondhand, and prenatal). While the variety in exposure types is informative and provides preliminary results, the relatively small sample sizes and limited number of studies overall make it clear that there is much more to be done in order to more definitively elucidate the effects of cigarette smoke (of any type) on ncRNA expression and the later downstream effects on behavioral health and other medical conditions.
The possible influence of epigenetic modification, including ncRNA expression, due to exposure to cigarette smoke of any kind (i.e., passive or secondhand, primary, or during pregnancy) on behavioral health and other medical conditions is manifold. As outlined above, the three best-characterized forms of ncRNA are microRNA (miRNA), piRNA, and long ncRNA, with miRNA garnering particularly great attention. In fact, at the time of the preparation of this review, there were virtually no published data on exposure-associated alterations to piRNA and relatively few studies on cigarette smoke-associated alterations to long ncRNA. Thus, unanswered questions include, but are not limited to, (i) the effects of cigarette smoke on piRNA and long ncRNA in multiple tissues, in animals and in humans, (ii) the effects of cigarette smoke on miRNA in the brain which might alter critical neurobehavioral circuitry in the developing brain or the adult brain, (iii) the influence of smoke exposure on ncRNA in asthma, lung cancer or other medical outcomes, and (iv) intergenerational transmission of smoking-related ncRNA changes (i.e., grandmaternal smoking influences which may affect germline cells and those epigenetic changes which may escape reprogramming during development). Use of animal models in mutually informative translational research (especially in inbred lines where genetic background is held constant) may further triangulate our ability as a field to investigate these issues (Knopik et al., Under Review).
It is clear, even from the relative paucity of research in this area, that ncRNA are biologically relevant and play an important role in the disease process. ncRNA alterations may also provide valuable information about therapeutic interventions. Yet, how do we align this information with the current state of science? One part of this answer lies in the research or clinical question of interest. If the question is whether cigarette smoke exposure, whether mainstream or sidestream, alters ncRNA expression and leads to the development of cancer cells, then the tissues of interest may indeed be relatively easily accessible. Animal models can be used to investigate ncRNA alterations in multiple tissues of interest, such as the lung, the esophagus, salivary glands, and bladder, but as with all model systems, consideration must be taken in study design regarding differential degrees of conservation of particular ncRNA across species. These preclinical models can be examined alongside human data, where biopsies of particular tissues may be available. However, if the outcome of interest is behavioral or psychiatric in nature (e.g., addiction, executive function, impulsive behavior, response to stress), the approach is less clear. Ideally, to consider the role of cigarette smoke exposure and ncRNA alterations on subsequent behavior, one would want to examine brain tissue. In humans, this is unavailable unless one considers post-mortem tissue; however, even then there is then the question of whether such investigations might be confounded due to potential ncRNA alterations associated with cause of death. There is considerable debate about the utility of blood as a biomarker for gene expression in brain and other tissues (Tsuang et al., 2005
; Tian et al., 2009
; Shivapurkar and Gazdar, 2010
; Kukreja et al., 2011
). In the search for a biomarker with clinical utility, blood does have certain advantages. Blood is an accessible tissue that can be relatively easily obtained, and while not a perfect representation of what might be expressed in brain, it can provide useful information for screening purposes. In either scenario, whether the health outcome is more psychological or somatic in nature, an additional question lies in what aspect of cigarette smoke leads to epigenetic alterations: more specifically, is it nicotine or one of the 4000+ other xenobiotics (e.g., foreign substances) found in cigarette smoke – or one of the multitude of complex mixtures of these xenobiotics – which are most responsible for leading to epigenetic alterations? Carefully examining this piece of the equation will also be key to developing a better understanding of how components of cigarette smoke alter ncRNA expression and function, but also the utility of using such cigarette smoke-modulated ncRNA in diagnostic and therapeutic interventions.
In summary, increasing attention to the study of ncRNA and to “environmental epigenetics” (Reamon-Buettner et al., 2008
) has inspired more researchers to embark on work to better understand how environmental exposures, such as cigarette smoke, affect ncRNA expression and function. Recommendations for future research include using both human cohorts and model systems to more comprehensively determine how the type, timing, frequency, duration, and degree of cigarette smoke exposure may alter miRNA, piRNA, and long ncRNA expression and function in a variety of tissues, thereby having the power to alter a number of health and developmental processes. The relative dearth of data demonstrating the effects of cigarette smoke on piRNA and relatively limited number of studies investigating the impact of cigarette smoke on long ncRNA underscore the need for future research to better describe such potentially hazardous effects of cigarette smoke on these two species of ncRNA and the processes they regulate. Hypothesis-generating approaches, such as microarray technology, when used in tandem with gold-standard validation approaches, such as Real-Time PCR, will be important for developing more agnostic study designs for discovering how ncRNA individually and collectively may be responsive to cigarette smoke exposure. Use of target prediction strategies combining in silico
target prediction analysis with empirical target prediction confirmation (i.e., via Western blot) will enable researchers to better streamline their efforts to discover currently unknown targets of miRNA. Tools for predicting potential targets of piRNA and long ncRNA remain in early development and such bioinformatic tools will prove especially useful for further determining the functions of piRNA and long ncRNA, especially in the context of harmful environmental exposures. This will not only enhance understanding of how these harmful exposures impact health but may suggest the utility of ncRNA as both therapeutic targets and biomarkers for determining treatment efficacy. Together, these advances will be crucial for determining how alterations to the expression and function of ncRNA may be important modes by which environmental exposures, such as cigarette smoke, influence health outcomes throughout the life course.