Analysis of MicroRNAs and their potential targets in human embryonic stem cell-derived neurons treated with the anesthetic propofol

Growing evidence demonstrates that prolonged exposure to general anesthetics, including propofol, induces widespread neuroapoptosis followed by long-term memory and learning disabilities in animal models. The underlying mechanisms of anesthetic-induced neurotoxicity are complex and not well understood. In addition, there is no direct clinical evidence of the detrimental effects of anesthetics in human fetuses, infants, or children. Development of an in vitro neurogenesis system of human stem cells opens up avenues of research for advancing our understanding of the issues of anesthetic-induced developmental neurotoxicity using a relevant human model. One avenue for investigating the mechanisms behind this neuroapoptosis is through evaluation of microRNA expression. MicroRNAs are endogenous, small, noncoding RNAs that negatively regulate target gene expression. MicroRNAs have been implicated to play important roles in many different disease processes, including neurological diseases. Our recent publication showed that among 84 microRNAs screened, propofol exposure altered the expression of 20 microRNAs in human embryonic stem cell (hESC)-derived neurons. Specifically, downregulation of microRNA-21 (miR-21) conferred by propofol played functional roles in the propofol-induced neurotoxicity. In this chapter, we outline (1) the protocol of human neuron differentiation from stem cells and (2) the protocols for analyzing microRNA expression (using our miR-21 study as an example) by quantitative reverse transcription-PCR and screening potential targets of miR-21 by Western blot assay following propofol exposure in stem cell-derived human neurons.