Fig. 6: HuR stabilizes BIRC3 mRNA depending on circNUP54-induced cytoplasmic export.

A, B BIRC3 mRNA and its encoded protein, cIAP2, were detected using RT-qPCR and WB in transient HuR knockdown or overexpression HCC cells, respectively. C RIP assay indicated BIRC3 mRNA was enriched by HuR. D RNA pull-down assay revealed HuR was pulled-down by the BIRC3 3’UTR probe. E Plasmids expressing FLAG-labeled FL or RRM-truncated HuR were transfected into Huh7 cells. The RIP products, precipitated using anti-FLAG, were subjected to qRT-PCR analysis to assess BIRC3 mRNA enrichment. F The 293T cells transfected with FL or RRM-truncated HuR plasmids were subjected to RNA pull-down using a BIRC3 3′UTR probe. WB detected the pulled-down HuR by anti-FLAG. G, H The remaining BIRC3 mRNA was detected using qRT-PCR in HuR up-regulated or down-regulated Huh7 cells after ActD treatment. I, J Degradation rate of BIRC3 mRNA in Huh7 cells transfected with indicated siRNAs or plasmids. K CircNUP54 overexpression or knockdown Huh7 cells were subjected to RIP assays precipitated by HuR. The enrichment of BIRC3 mRNA was analyzed using qRT-PCR. L BIRC3 3’UTR probe pull-down was performed in Huh7 cells with circNUP54 overexpression or knockdown. The pulled down HuR was detected using WB. M, N BIRC3 mRNA and cIAP2 were determined using RT-qPCR and WB in HCC cells transfected with wild-type or mutant plasmids of circNUP54. O IF images of HuR (green) in circNUP54 knockdown or overexpression HCC cells. CircNUP54 overexpression promotes the cytosolic accumulation of HuR; Scale bar = 10 μm. P, Q Quantitative WB determining the HuR levels in total lysates or subcellular fractions of the circNUP54 overexpression or knockdown HCC cells. Cytosolic HuR was increased by circNUP54 overexpression. Data presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).