130P - Primary cilium (PC) associated BBSome gene expression profile during oxidative stress in 2D and 3D culture models of mesothelial cells

Background

PC is a sensory organelle that is subject to environmental cues such as oxidative stress. The PC is composed of Bardet-Biedl family of proteins that form the BBSome complex and facilitate cell surface receptor engagement and trafficking. Mesothelial cells that line the pleural cavity are sensitive to native pleural homeostasis. Pleural effusion formation is usually accompanied by high oxidative stress and it is unknown whether the status of the PC BBSome changes as a result in mesothelial cells in 2D or 3D. We sought to assess BBsome component gene expression in 2D and 3D in-vitro models of oxidative stress using 100mM H₂O₂.

Methods

Synchronized benign mesothelial cells, MeT-5A, were cultured on 2D planar surfaces on in a hanging drop conformation using standard culture medium or media with 100mM H₂O₂ for 24 hours. Cells were lysed for RNA processing and cDNA was prepared followed by qPCR quantitation with gene specific primers for BBS1, BBS2, BBS4, BBS5, BBS7, BBS9 and BBS18 and b-actin. Log 2 transformed 2ˆ^{-DD Ct} values were used to compare relative gene expression.

Results

BBS1, 2, 4, 7, 9, 18 gene expression did not change in a 2D culture model after 100mM H₂O₂ exposure. BBS5 expression was undetectable. BBS5 expression was observed in 3D irrespective of 100mM H₂O₂ stimulus. There were no significant changes to any BBS genes studied in 3D with or without 100mM H₂O₂ stimulus. BBS1, BBS2 and BBS4, BBS5, BBS9 and BBS18 expression in 3D were confined to the negative values as opposed to positive in 2D conditions during 100mM H₂O₂ stimulation.

Conclusions

Oxidative stress does not induce changes to any BBS gene expression in 2D cultures or 3D culture configuration. Expression patterns differ qualitatively between 2D and 3D cell culture and this warrants further investigation.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Hellenic Foundation for Research & Innovation.

Disclosure

All authors have declared no conflicts of interest.