Multiple circRNAs regulated by QKI5 conjointly spongemiR-214-3p to antagonize bisphenol A-inducedspermatocyte toxicity: Cooperative effects of circRNAs on spermatocyte toxicity

Although circular RNAs (circRNAs) are found to play important roles in many pathophysiological processes, the canonical theory that they act as microRNA sponges is now more and more challenged, given that most circRNAs only have few binding sites in a particular microRNA. Our previous study revealed that some up-regulated circRNAs play protective roles in bisphenol A (BPA)-induced toxicity in GC-2 germ cells. Here by CCK-8 assay, apoptosis assay, qRT-PCR and western blot analysis, we further discover that circRNAs (represented by circDcbld2, circMapk1 and circTbcld20) can cooperatively sponge miR-214-3p and then up-regulate AKT1 in ameliorating BPA-induced reproductive toxicity. They share binding sites with miR-214-3p and collectively reinforce the sponging effects. In addition, the upstream regulation mechanism, proven by bioinformatics analysis and in vitro gain- and loss-of-function study, shows that down-regulation of RNA binding protein QKI5 after BPA exposure can increase the expressions of these protective circRNAs, and thus activate the cell protective process. The QKI5-circDcbld2/ circMapk1/circTblcd20-miR-214-3p-AKT1 axis ameliorates the toxic effect of BPA on GC-2 cells. Many other circRNAs up-regulated upon BPA treatment and QKI5 down-regulation also show binding sites with miR-214-3p. Thus the above axis may also be extrapolated to other circRNAs. Our results enrich the context of circRNA sponge mode and may provide new ideas in future multiple nucleic acid therapy.