Fig. 4: CircNUP54 knockdown inactivates the NF-κB pathway via downregulation of BIRC3.

A Clustered heat map of RNA transcriptome sequencing in Huh7 cells transfected with sh-NC or sh-circNUP54. B, C KEGG and GO enrichment analyses of downregulated genes after circNUP54 knockdown. D–G qRT-PCR of 68 HCC samples verified the correlation of circNUP54 with four downstream target genes, selected by overlapping the top 20 most significantly downregulated genes and NF-κB pathway-involved genes. H, I qRT-PCR was performed to determine the expression of BIRC3, LTB, AKT3, and CXCL3 in Huh7 and SNU449 cells with circNUP54 knockdown. J BIRC3 expression in HCC and normal liver tissue (TCGA database). K WB analysis of cIAP2, a protein encoded by BIRC3 mRNA, in Huh7 and SNU449 cells with circNUP54 knockdown. GAPDH served as an internal reference. L WB and quantitative analysis of the NF-κB pathway-related proteins (p-p65, p65, IκBα, and p-IκBα) in indicated HCC cells after circNUP54 knockdown. M The cIAP2 expression of Huh7 cells after transfection with siRNAs (si-NC and si-BIRC3) or plasmids (Vector or BIRC3). Tubulin was used as an internal control. N WB and quantification results of p-p65/t-p65 and p-IκBα/t-IκBα in Huh7 cells, showing that knockdown of BIRC3 impaired the activation of the NF-κB pathway caused by circNUP54 overexpression. O Overexpression of BIRC3 rescued the inhibitory effect of circNUP54 knockdown on the NF-κB pathway in Huh7 cells. Data presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (Student’s t-test).