EIF4A3-induced circ_0084615 contributes to the progression of colorectal cancer via miR-599/ONECUT2 pathway

As we observed in Fig. 1 A-C, volcano plot showed the differentially expressed circRNAs in databases GSE126094, GSE138589 and GSE142837. Among these, 4 aberrantly expressed circRNAs (hsa_circ_0000512, hsa_circ_0000467, hsa_circ_0040809 and hsa_circ_0084615) were simultaneously found in GSE126094, GSE138589 and GSE142837 (Fig. 1D). Heatmap analysis showed that hsa_circ_0000512, hsa_circ_0000467, hsa_circ_0040809 and hsa_circ_0084615 were all upregulated in CRC tissues compared to normal tissues (Fig. 1E-G). qRT-PCR showed that hsa_circ_0000512, hsa_circ_0000467, hsa_circ_0040809 and hsa_circ_0084615 were highly expressed in CRC tissues (n = 8) compared to corresponding control tissues (n = 8) (Fig. 1 H). Given the higher expression of hsa_circ_0084615 than hsa_circ_0000512, hsa_circ_0000467 and hsa_circ_0040809 in CRC tissues, circ_0084615 was selected for further study. Compared to normal tissues (n = 54), circ_0084615 was markedly elevated in CRC tissues (n = 54) (Fig. 1I). Moreover, our results showed that circ_0084615 level was increased in CRC patients in TNM stage III (n = 25), lymph node metastasis (n = 22) and poor differentiation (n = 20) groups compared to the patients in TNM stages I + II (n = 29), not lymph node metastasis (n = 32) and well/moderate differentiation (n = 34) groups (Fig. 1 J-L). Besides, the patients were divided into 2 groups (high level of circ_0084615 and low level of circ_0084615) according to the median value and high level of circ_0084615 was related to poor overall survival of CRC patients (Fig. 1 M).

As shown in Fig. 2A, circ_0084615 was notably increased in SW620, DLD1, SW480 and HT-29 cells compared to FHC cells. Circ_0084615 located at chr8: 62593526–62596747 and was formed by the exons 2–4 of ASPH with a mature length of 264 nt (Fig. 2B). Then, divergent primers were designed to amplify circ_0084615 and convergent primers were designed to amplify ASPH. We found that circ_0084615 amplification product was only detectable in cDNA by divergent primers and ASPH was amplified in both cDNA and genomic DNA (gDNA) by convergent primers (Fig. 2 C). Due to the lack of 3’ poly a tail, we analyzed the existence of circ_0084615 in the reverse transcription products by using random hexamer primers and oligo (dT)18 primers. We found that oligo (dT)18 primers barely amplified circ_0084615 (Fig. 2D and E). Act D assay showed that circ_0084615 possessed a longer half-life than ASPH (Fig. 2 F and G). RNase R assay showed that ASPH was digested by RNase R, while circ_0084615 was resistant to RNase R (Fig. 2 H and I). In addition, it was found that circ_0084615 mainly enriched in the cytoplasm instead of the nucleus in SE620 and DLD-1 cells (Fig. 2 J and K).

To clarify the potential roles of circ_0084615 in CRC progression, loss-of-function study was conducted by transfecting sh-circ_0084615 into SW620 and DLD-1 cells. As a result, sh-circ_0084615 transfection led to a distinct reduction in circ_0084615 expression in SW620 and DLD-1 cells compared to sh-NC control groups (Fig. 3 A). EdU assay and colony formation assay showed that circ_0084615 silencing markedly suppressed the proliferation and colony formation of SW620 and DLD-1 cells compared to sh-NC groups (Fig. 3B and C). As demonstrated by wound healing assay and transwell assay, the migration and invasion of SW620 and DLD-1 cells were restrained by decreasing circ_0084615 in comparison with sh-NC control groups (Fig. 3D and E). Tube formation assay indicated that circ_0084615 knockdown repressed the angiogenesis ability of HUVECs compared to control groups (Fig. 3 F). Besides, the protein levels of epithelial-mesenchymal transition (EMT)-related markers (ZEB2, E-cadherin and Vimentin) and VEGFA were measured. The results presented that circ_0084615 deficiency decreased ZEB2, Vimentin and VEGFA protein levels and increased E-cadherin protein level in SW620 and DLD-1 cells (Fig. 3G and H). Collectively, circ_0084615 silencing inhibited CRC cell growth, metastasis and angiogenesis.

Through analyzing starbase (http://​starbase.​sysu.​edu.​cn/​starbase2/​) and circinteractome (https://​circinteractome.​irp.​nia.​nih.​gov/​), miR-599 was found to be the target of circ_0084615 (Fig. 4 A). RIP assay indicated that circ_0084615 and miR-599 were significantly enriched in the immunoprecipitated complexes of anti-Ago2 groups compared to anti-IgG groups (Fig. 4B and C). RNA pull-down assay showed that bio-miR-599-wt pull down more circ_0084615 than bio-NC and bio-miR-599-mut control groups (Fig. 4D and E). The binding sites between circ_0084615 and miR-599 were exhibited in Fig. 4 F. MiR-599 mimic transfection led to an elevation of miR-599 expression in SW620 and DLD-1 cells (Fig. 4G). Dual-luciferase reporter assay showed that the luciferase intensity of circ_0084615-wt was repressed in SW620 and DLD-1 cells with miR-599 overexpression, but the luciferase intensity of circ_0084615-mut was not changed (Fig. 4 H and I). Expectedly, miR-599 was lowly expressed in CRC tissues and cells relative to normal tissues and cells (Fig. 4 J and K). All these results indicated that miR-599 was a target of circ_0084615.

As presented in Fig. 5 A, anti-miR-599 transfection reduced miR-599 level in SW620 and DLD-1 cells compared to anti-miR-599. Then sh-NC + anti-NC, sh-circ_0084615 + anti-NC or sh-circ_0084615 + anti-miR-599 was transfected into SW620 and DLD-1 cells to explore the relationship between circ_0084615 and miR-599 in regulating CRC progression. EdU assay and colony formation assay showed that miR-599 inhibition reversed circ_0084615 knockdown-mediated suppressive effects on cell proliferation and colony formation in SW620 and DLD-1 cells (Fig. 5B and C). Wound-healing assay and transwell assay indicated that circ_0084615 silencing repressed the migration and invasion of SW620 and DLD-1 cells, while these effects were partially ameliorated by reducing miR-599 (Fig. 5D and E). Tube formation assay showed that circ_0084615 interference hampered the tube formation ability of HUVECs, with miR-599 downregulation abated the effect (Fig. 5 F). Furthermore, the effects of circ_0084615 knockdown on ZEB2, E-cadherin, Vimentin and VEGFA levels in SW620 and DLD-1 cells were reversed by decreasing miR-599 (Fig. 5G and H). Taken together, circ_0084615 knockdown inhibited the malignancy of CRC cells by targeting miR-599.

Through analyzing GSE12609, we selected 100 upregulated genes in CRC and found that only ONECUT2 was simultaneously predicted to be the target gene of miR-599 by starbase and miRDB (http://​mirdb.​org/​) (Fig. 6 A). RIP assay showed that the enrichment of ONECUT2 and miR-599 was increased by anti-Ago2 RIP compared to anti-IgG RIP groups (Fig. 6B and C). RNA pull down assay showed that ONECUT2 was enriched by bio-miR-599-wt compared to bio-NC or bio-miR-599-mut (Fig. 6D and E). As shown in Fig. 6 F, ONECUT2 contained miR-599 binding sites. Then dual-luciferase reporter assay indicated that miR-599 elevation repressed the luciferase activity of ONECUT2 3’UTR-wt in SW620 and DLD-1 cells but did not affect the luciferase activity of ONECUT2 3’UTR-mut (Fig. 6G and H). These results demonstrated the interaction between ONECUT2 and miR-599. Moreover, miR-599 overexpression markedly decreased the mRNA and protein levels of ONECUT2 in both SW620 and DLD-1 cells (Fig. 6I and J). Circ_0084615 knockdown decreased the mRNA and protein levels of ONECUT2 in SW620 and DLD-1 cells, whereas miR-599 inhibition reversed the impacts (Fig. 6 K and L). Indeed, qRT-PCR and IHC assay showed that ONECUT2 level was increased in tumor tissues compared to normal tissues (Fig. 6 M and N). In addition, ONECUT2 mRNA and protein levels were increased in CRC cells compared to FHC cells (Fig. 6O and P). Collectively, circ_0084615 altered ONECUT2 expression by targeting miR-599.

The transfection of ONECUT2 overexpression resulted in a marked elevation of ONECUT2 protein level in SW620 and DLD-1 cells (Fig. 7 A). Next, whether miR-599 could regulate CRC cell progression by targeting ONECUT2 was investigated. As illustrated by EdU assay and colony formation assay, miR-599 overexpression restrained the ability of SW620 and DLD-1 cells to proliferate, while ONECUT2 elevation rescued the effect (Fig. 7B and C). Overexpression of miR-599 impeded the migration and invasion of SW620 and DLD-1 cells, with ONECUT2 upregulation reversed the effects (Fig. 7D and E). Tube formation assay showed that miR-599 overexpression inhibited the tube formation ability of HUVECs, while the impact was weakened by elevating ONECUT2 (Fig. 7 F). Additionally, miR-599 enhancement reduced ZEB2, Vimentin and VEGFA levels and elevated E-cadherin levels in SW620 and DLD-1 cells, with ONECUT2 overexpression abated the impacts (Fig. 7G and H). To sum up, miR-599 overexpression inhibited the malignant behaviors of CRC cells by targeting ONECUT2.

It has been reported that some RNA-binding proteins can bind to the flanking of the intron sequence of circRNAs and influence circRNA production [19]. EIF4A3 is a regulator of exon splicing [20]. Through analyzing circinteractome (https://​circinteractome.​irp.​nia.​nih.​gov/​), we found that EIF4A3 had 5 binding sites on the upstream region of ASPH mRNA transcript (Fig. 8 A). RIP assay showed that EIF4A3 could bind to ASPH mRNA via the binding regions a and b but not circ_0084615 (region c) (Fig. 8B-D). These results indicated that EIF4A3 could bind to the flanking sequences of circ_0084615 through the potential binding sites. Furthermore, sh-EIF4A3 or oe-EIF4A3 was transfected into SW620 and DLD-1 cells to reduce or elevate EIF4A3 expression, which were verified by western blot assay (Fig. 8E and F). Of note, our results showed that EIF4A3 knockdown reduced circ_0084615 level and EIF4A3 overexpression increased circ_0084615 expression in SW620 and DLD-1 cells (Fig. 8G and H). Taken together, EIF4A3 positively regulated circ_0084615 expression.

Subsequently, the roles of circ_0084615 in tumor growth were investigated. The results of murine xenograft model assay showed that the mice with circ_0084615 silencing possessed the reduced tumor volume and tumor weight compared to control groups (Fig. 9 A-C). IHC assay showed that ONECUT2, ZEB2, Vimentin and VEGFA levels were reduced and E-cadherin level was elevated in the xenograft tumors in sh-circ_0084615 groups compared to sh-NC groups (Fig. 9D). These results indicated that circ_0084615 played a positive role in tumor growth in vivo.