Hypoxia-induced circPLOD2a/b promote migration and invasion of GBM cells via suppressing XIRP1 through binding to HuR [2]
Background. Glioblastoma (GBM) is the most lethal form of brain tumors in human adults, and hypoxia is a common microenvironment in this disease. Circular RNAs (circRNAs) are identified as regulators in cancers including GBM. However, the specific roles of circRNAs in GBM under hypoxic condition remains elusive. Methods. RNA-seq was employed to investigate the expression profile of circRNAs in U87 cells under normoxia and hypoxia. Two circRNAs spliced from the same parental gene, named as circPLOD2a and circPLOD2b, were identified as hypoxia-responsive circRNAs, whose circular structures were verified by, qRT-PCR, RNase R digesting and Sanger sequencing in U87. Knockdown and overexpression of circPLOD2a/b in GBM cells were used to investigate the biological functions of these two circRNAs on tumor progression. Fluorescence in situ hybridization, RNA-pull down, mass spectrometry and RNA immunoprecipitation assays were conducted to explore the interactions between circPLOD2a/b, human antigen R (HuR), xin actin binding repeat containing 1 (XIRP1). Results. CircPLOD2a/b were significantly up-regulated in hypoxic GBM cells and directly induced by HIF1a under hypoxia. Overexpression of circPLOD2a/b enhance GBM cell invasion and migration, which could be inhibited by circPOD2a/b knockdown. Mechanistically, circPLOD2a/b competitively bind to HuR, inhibiting the interaction between HuR and XIRP1, thus promoting the tumor aggressiveness in GBM both in vitro and in vivo through down-regulating XIRP1.  Conclusions. Hypoxia-induced circPLOD2a/b were identified as key regulators of migration and invasion in GBM cells, and act as oncogenic circRNAs to attenuate the interaction between HuR and XIRP1, which may be potential therapeutic strategy for GBM treatment. Overall design: Human GBM cell U87-wt were planted in six 6-cm dish. Three dishes of cells were cultured in hypoxia (1% O2, 94% N2 and 5% CO2) and another three dishes treated in normoxia  (21% O2, 74% N2 and 5% CO2) respectively. 24 hours later, the DMEM medium was removed, and 1mL PBS buffer was added into each dish to wash medium residue. Remove all the PBS buffer from the dishes and add 1mL Trizol buffer. Solution from each dish was collected into 1.5mL EP tube, and stored in liguid nitrogon for further analysis.