CircEZH2 inhibits opening of mitochondrial permeability transition pore via interacting with PiC and up-regulating RSAD2

Abstract

Transmissible gastroenteritis virus (TGEV) infection can lead to mitochondrial damage in porcine intestinal epithelial cells-jejunum 2 (IPEC-J2) cell line. The abnormal opening of mitochondrial permeability transition pore (mPTP) is the most important factor for mitochondrial damage. We previously demonstrated that circEZH2 could inhibit the abnormal opening of mPTP by binding miR-22. However, circEZH2 binding to miR-22 cannot completely enable mPTP opening to recover to normal level compared with the control group. So, we assume that circEZH2 also regulates the mPTP opening in other ways. To prove it, we identified the differentially expressed proteins (DEPs) caused by circEZH2 and circEZH2-interacting proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It turns out there are 123 DEPs (0.83 ≤ fold change ≥ 1.2) upon overexpression circEZH2 and 200 proteins interacted with circEZH2. The kyoto encyclopedia of genes and genomes (KEGG) analysis, gene ontology (GO) analysis, subcellular localization analysis, and protein interaction network results show that the DEPs and circEZH2-interacting proteins may involve in the regulation of mPTP opening. RNA immunoprecipitation (RIP) assay and flow cytometry (FCM) results indicate that circEZH2 can inhibit the opening of mPTP by interacting with Pi carrier (PiC, also named SLC25A3). Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and FCM results reveal that circEZH2 can inhibit mPTP opening by promoting the expression of radical s-adenosyl methionine domain-containing protein 2 (RSAD2). In addition, PiC can promote RSAD2 expression. The data indicate that circEZH2 inhibits TGEV-induced mPTP opening by interacting with PiC and upregulating RSAD2.