Investigating the role of microRNAs on the transgenerational effects of nicotine using Caenorhabditis elegans

Abstract

This project aims to investigate the effects of nicotine, the highly addictive and toxic substance in tobacco-based products, has on offspring (1st and 2nd generations, F1 and F2) using the model organism Caenorhabditis elegans (C. elegans). C. elegans are microscopic nematode worms that are used as a model organism to study epigenetic effects passed down through generations. Due to their transparency, small size, and cellular simplicity, researchers can easily observe the internal structures and processes of C. elegans. To characterize nicotine-dependence, our lab looks at three behaviors: stimulation, adaption, and withdrawal. We hypothesize that maternal nicotine exposure (F0 generation) increases susceptibility to nicotine dependence in F1 and F2 generations. To test this, F0 worms were exposed to a 61.7 µM nicotine solution or a vehicle control (K medium) at the L3 larval stage for 24 hours, then the behaviors were observed in F0, F1, and F2 generations, where the F1 and F2 generations were not exposed to nicotine. Locomotion behaviors were analyzed using WormLab. For nicotine-exposed worms, an increased speed in a nicotine-free environment indicates withdrawal, while a lower locomotion speed in nicotine-containing environment indicates nicotine adaption. To explore miRNA-mediated molecular mechanisms, RNA interference with the miRNA inhibitor anti-cel-miR-1 will knock down miR-1, which regulates nicotinic acetylcholine receptor (nAChR) expression. Behavioral changes in miRNA inhibitor and nicotine co-treated worms will be compared to nicotine-only treated worms and vehicle controls. Statistical analyses of locomotion behaviors revealed transgenerational inheritance of nicotine-dependent behaviors and the important role of miRNA-1 in nicotine-dependent behaviors. With miR-1 inhibition in F0 generation, the nicotine-dependent behavior was cured in F0 and the following F1 and F2 generations. This represents an important finding in the mechanism of nicotine-dependent behaviors and potentially applies to the development of future miRNA-mediated therapeutic strategies.