Figure 6.

EIF4A3 Induced circ-BNIP3 Expression in H9c2 Cells

(A) The binding sites for EIF4A3 on the BNIP3 mRNA transcript at the upstream region of circ-BNIP3 were obtained from Circular RNA Interactome. (B) Pull-down assay confirmed the enrichment of EIF4A3 protein in BNIP3 mRNA pull-down. (C) Quantitative real-time PCR following the EIF4A3-RIP assay confirmed the binding of EIF4A3 on the predicted binding region (a, b, c, and d) of BNIP3 mRNA. H19, a known interacting long noncoding RNA (lncRNA) with EIF4A3, was the positive control. The intron 1 of BNIP3 mRNA was used as negative control. (D) The schematic diagram of 5 RNA constructs with EIF4A3 binding sites, which were truncated to different degrees (s1–s5). Laz and H19 were the negative control and positive control. The pull-down assay was conducted to analyze the interplay between EIF4A3 and BNIP3 mRNA (s1–s5). (E) The overexpression and knockdown of EIF4A3 in H9c2 cells were confirmed by quantitative real-time PCR analysis. (F) The expression of circ-BNIP3 under EIF4A3 overexpression in H9c2 cells treated with or without hypoxia was analyzed upon quantitative real-time PCR analysis. Bar graphs were shown as mean ± SD. *p < 0.05, **p < 0.01. NS: no significance.