Image_2_NFIX Circular RNA Promotes Glioma Progression by Regulating miR-34a-5p via Notch Signaling Pathway.TIF

Objective: The present study aimed to explore the association between NFIX circular RNA (circNFIX) and miR-34a-5p in glioma. Furthermore, this study investigated the influence that circNFIX has on glioma progression through the upregulation of NOTCH1 via the Notch signaling pathway by sponging miR-34a-5p.

Methods: We applied five methods, CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2, to screen for circRNAs with differential expression between three glioma tissue samples and three paired normal tissue samples. The GSEA software was used to confirm whether significantly different pathways were activated or inactivated in glioma tissues. The binding sites between circNFIX and miR-34a-5p were confirmed by TargetScan. QRT-PCR and western blot were used to measure the relative expression levels of circNFIX, miR-34a-5p and NOTCH and identify their correlation in glioma. RNA immunoprecipitation (RIP) validated the binding relationship between circNFIX and miR-34a-5p, while the targeted relationship between NOTCH1 and miR-34a-5p was verified by a dual luciferase reporter assay. Cell viability and mobility were examined by a CCK-8 assay and wound healing assay, and a flow cytometry assay was employed to analyze cell apoptosis. The nude mouse transplantation tumor experiment verified that si-circNFIX exerted a suppressive effect on glioma progression in vivo.

Results: Twelve circRNAs were differentially expressed between the tissue types. Of those, circNFIX was the sole circRNA to be overexpressed in glioma among the five methods of finding circRNAs. In addition, the Notch signaling pathway was considerably upregulated in tumor tissues compared with the paired normal brain tissues. It was determined that circNFIX acted as a sponge of miR-34a-5p, a miRNA that targeted NOTCH1. Downregulation of circNFIX and upregulation of miR-34a-5p both inhibited cell propagation and migration. Furthermore, a miR-34a-5p inhibitor neutralized the suppressive effect of si-circNFIX on glioma cells. Si-circNFIX and miR-34a-5p mimics promoted cell apoptosis. Moreover, it was demonstrated in vivo that si-circNFIX could suppress glioma growth by regulating miR-34a-5p and NOTCH1.

Conclusion: CircNFIX was markedly upregulated in glioma cells. CircNFIX could regulate NOTCH1 and the Notch signaling pathway to promote glioma progression by sponging miR-34a-5p via the Notch signaling pathway. This finding provided a deeper insight into the function of circNFIX in human glioma cancer progression.