CircERBB2 Regulates Ribosomal DNA Transcription Associated With Gallbladder Cancer Growth

Objectives Gallbladder cancer(GBC)is a malignancy of biliary tract with very unfavored prognosis.Circular RNAs were found to play essential roles through different cancer types.To amplify on molecular mechanism of GBC biology and improve prognosis of GBC,we performed circular RNA expression profiling of GBC.We wanted to seek out circular RNAs that affect GBC biology.Methods RNA sequencing was performed on 10 primary GBC samples and the respective para-tumor tissues to reveal m RNA,mi RNA,lnc RNA and circular RNA expression profile.Global changes of circular RNAs during GBC formation were analyzed.We found a circular RNA named circ ERBB2 that increased significantly during GBC formation.circ ERBB2 was effectively knockdown with si RNAs and CRISPR-cas9 technology,with no detectable effects on homogenous m RNA ERBB2.Moreover,circ ERBB2 over expression plasmid was constructed for further conformation of circ ERBB2's effects on GBC biology.Subcellular localization of circ ERBB2 was clarified by FISH.q PCR and dual luciferase reporter assay were performed to identify circ ERBB2's any measurable effect on ribosomal RNA(r RNA)biogenesis.RNA pulldown assay was performed to identify circ ERBB2-associated proteins.RNA immunoprecipitation was performed for further confirmation of the interaction between circ ERBB2 and the protein.Western blot,IF,etc.were performed to decipher circ ERBB2's effects on the associated protein.A tissue microarray contains 149 primary GBC tissues was used to analysis if circ ERBB2 was associated with prognosis of GBCpatient.Results Circular RNA expression changed significantly during GBC genesis.Total circular RNA expression decreased significantly in GBC tissues.Meanwhile,linear association between circular RNAs and homogenous m RNA increased significantly in cancer tissue.We found circ ERBB2,which was a circular RNA produced from back-splicing between exon 3 and exon 7 of ERBB2 gene,increased significantly in GBC.We found that proliferation of GBC cells decreased significantly when circ ERBB2 decreased.And over expression of circ ERBB2 promotes GBC growth,in vitro and in vivo.circ ERBB2 was found to accumulate in the nucleolus and promote ribosomal DNA(r DNA)transcription.A set of potential circ ERBB2 associated proteins were identified with RNA pulldown.Among them was PA2G4,which is associated with r DNA transcription and cell proliferation.Interaction between circ ERBB2 and PA2G4 was further confirmed by RNA immunoprecipitation.We found that circ ERBB2 promotes nucleolar localization of PA2G4.When translocated to nucleolus,PA2G4 interactes with TIFIA,promotes r DNA transcription and accelerates GBC cell growth.Increased expression of circ ERBB2 is associated with worse prognosis of GBC patient(p<0.05).Conclusions Circular RNA expression profile changed significantly during GBC genesis.circ ERBB2 increased significantly in GBC.circ ERBB2 acceralates GBC growth by promoting r DNA transcription.circ ERBB2-PA2G4-TIFIA axis plays critical role in regulating Pol I activity and cellular proliferation.circ ERBB2 is associated with worse prognosis of GBC patients.