CircURI1 Inhibits Metastasis Via Modulating Alternative Splicing In Gastric Cancer

Gastric cancer(GC),an aggressive malignance,is the third leading cause of cancer-related mortality,largely caused by the presence of metastatic spread worldwide.Circular RNAs(cireRNAs)are covalent closed single-stranded endogenous RNA molecules,which have emerged as key regulators of human cancers;nevertheless the fundamental roles and the underlying mechanisms of circRNAs in GC remain mysterious.Here,we perform circRNA profiling of five-paired human gastric and the corresponding adjacent non-tumor(paraGC)specimens and characterize 245 significantly dysregulated circRNAs(152 downregulated and 93 upregulated)in GC.Then,integrative analysis of the previously reported GC circRNA profiles and our RNA-seq dataset identifies circURI1 back-spliced from exons three and four of unconventional prefoldin RPB5 interactor 1(URI1)as the upregulated candidate in GC.Realtime quantitative PCR(RT-qPCR)of 69-paired GC and paraGC tissues and fluorescence in situ hybridization(FISH)arrays demonstrate that circURIl exhibits the remarkably higher expression in GC compared with paraGC.The increased circURIl expression levels are significantly associated with early TNM stage(I-II)tumors and non-metastasis in GC patients.Depletion of circURIl with short interfering RNAs(siRNAs),antisense oligonucleotides(ASOs)or clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)results in the promotion of GC cell migration and invasion in vitro.In contrast,the observed pro-metastatic phonotype of circURIl ablation could be rescued by the ectopic overexpression of circURI1.Moreover,in vivo observations perform that shRNA-mediated repression of circURI1 leads to the increased and large lung metastases in a tail vein injection mouse model.RNA pulldown followed by mass spectrometry(MS)and RNA immunoprecipitation(RIP)assays confirm the direct interaction between circURI1 and heterogeneous nuclear ribonucleoprotein M(hnRNPM).Individual-nucleotide resolution CLIP(iCLIP)followed with RNA-sequencing(RNA-seq)of hnRNPM determines a 19-nt sequence in circURI1(GGCUGUAGGUUUAGUUGAG),which is proximal to the junction site of circURI1,as the hnRNPM binding site.Mechanistically,circURI1 interacts with hnRNPM to modulate alternative splicing of genes,involved in the process of cell migration,thus suppressing GC metastasis.Vascular endothelial growth factor A(VEGFA)is a functional target of circURI1 and circURI1 could promote exon 7 inclusion of VEGFA(VEGFAe7IN).Furthermore,Kaplan-Meier survival curve analysis of TCGA database reveals that the overall survival is obviously longer in GC patients with high VEGFAe7IN compared to those with low VEGFAe7IN.Finally,VEGFAe7IN isoform possesses a greater ability to recover the promotive effect of circURI1 knockdown on GC cell invasion than VEGFAe7EX.Collectively,our study expands the current knowledge regarding the molecular mechanism of circRNA-mediated cancer metastasis via modulating alternative splicing.