Chemoprevention of cigarette smoke-induced alterations of MicroRNA expression in rat lungs

Abstract

We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-kappaB pathway, transforming growth factor-related stress response, and angiogenesis. Some microRNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents.

Fig. 1

Scatterplots relating the expression of 484 miRNAs in the lung of rats treated with various chemopreventive regimens to the expression of the same miRNAs in the lung of rats, either Sham-exposed (top) or ECS-exposed (bottom). Central diagonal line in each panel, equivalence of expression between the groups compared on the X and Y scales; two outer diagonal lines in each panel, 2-fold variation intervals.

Fig. 2

Hierarchical cluster analysis linking the expression profiles of 484 miRNAs among variously treated experimental groups. Each line within each column, the intensity of expression of each miRNA on a color scale, from blue (lowest) to red (highest).

Fig. 3

Bidimensional PCA comparing the expression profile of 484 miRNAs among variously treated experimental groups.

Fig. 4

Real-time qPCR amplification curves (top) and positivity threshold (bottom) of let-7c expression in the lung of variously treated rats.