Effects of Melatonin on Caco-2 cells with a Synchronized Circadian Rhythm

The circadian rhythm is a 24-hour cycle that regulates physiological and mental processes across organisms. In peripheral tissues, the rhythm is regulated by so-called clock genes, such as BMAL1 and PER2, which maintain the synchronization through feedback loops. Almost half of human genes are regulated by clock gene expression. Melatonin, a metabolite produced by gut microbiota, has previously been shown to affect gene expression, as well as supporting the sleep-wake cycle and regulating immune responses. In vitro cell cultures often involve unsynchronized cells, which do not follow the same rhythm, they can however, be synchronized to produce a more physiologically relevant model. In this study, Caco-2 cells were synchronized using dexamethasone, and were thereafter used to investigate melatonin's effect on clock gene expression of BMAL1 and PER2. Gene expression analysis of the target genes and housekeeping genes GAPDH and RPLP0 was performed using qPCR. The results were inconclusive regarding melatonin's effect on clock gene expression. Further analyses revealed instead that exposure to melatonin caused a significant increase in variance of housekeeping genes. These findings highlight the importance of housekeeping gene validation and I provide suggestions for a solution applicable in future similar research; Performing validation of housekeeping genes in the presence of intended exposures, as well as on the intended cell line for greater reliability.