Fig. 3

EIF4A3 suppresses circPTEN1 expression. A. Left, identification of proteins pulled down by circPTEN1 upstream or downstream sequences with protein extracts from LoVo cells. The arrow indicates the additional band containing eIF4A3. Right, immunoblot analysis of eIF4A3 after pull-down assay showing its specific association with circPTEN1 upstream flanking sequence (upper panel); the enriched RNA labeled with biotin was evaluated by RT-PCR (lower panel). B. The binding sites of eIF4A3 were predicted in the flanking region of circPTEN1 using CircInteractome. C. The RIP assay was performed to verify the binding sites of eIF4A3 on circPTEN1 upstream sequences. H19 lncRNA was used as the positive control. D. The RNA pull-down assay was performed to analyze the interaction between eIF4A3 and several circPTEN1 upstream truncations (a1-a6). Laz was a nonsense sequence used as the negative control, and H19 was used as the positive control. E. qRT-PCR assay showing the relative levels of eIF4A3 (normalized to β-actin) in the peritumor and tumor tissues of colon cancer (n = 60). ***, P < 0.001. F. The correlation between eIF4A3 and circPTEN1 (n = 60). G. LoVo cells were infected with lentivirus expressing eIF4A3 shRNA, or scramble shRNA. A rescue experiment was conducted by infecting eIF4A3-knockdown cells with pLV-eIF4A3. The expression of eIF4A3 was validated by western blot, and the ratio of the grayscale value of the eIF4A3 band to the grayscale value of the corresponding GAPDH band was labeled (lower panel). The circPTEN1 level was evaluated by qRT-PCR (upper panel)