Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells

Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For thisreason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinalepithelial barrier and their response to altered gravity conditions was investigated, especially onthe protein level. In this study, we combined label-free shotgun proteomics and bioluminescentreporter gene assays to identify key proteins and pathways involved in the response of Caco-2cells under reference and microgravity conditions. A two-dimensional clinostat was modifiedwith 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteomeanalysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation,cellular and metabolic processes and localization. Bioluminescent reporter gene assays werecarried out to investigate microgavity-induced alterations on the transcriptional regulation ofkey targets, such as NF-kB pathway and CYP27A1. While no significant difference was foundin the basal transcription, a lower NF-kB basal activation in simulated microgravity conditionswas reported, corroborating the hypothesis of reduced immunity in microgravity conditions.