3.1. circMYO1C was a METTL3-induced circRNA and up-regulated in PDAC
To investigate the potential circRNAs regulated by m6A modification, we chose METTL3, a crucial m6A methyltransferase, to construct the overexpression of METTL3 and its control in PANC-1 cells. CircRNA microarray analysis revealed that numerous circRNAs were dys-regulated and we focused on an up-regulated circRNA (circMYO1C). CircMYO1C was a 186 bp length transcripts derived from the exon9-exon8 of MYO1C gene. Its ID was hsa_circ_0041234 (circBase) and hsa_circRNA_101936 (ArrayStar, Aksomics) (Figure 1A). Moreover, several candidate circRNAs were quantificationally validated by RT-PCR, and results demonstrated that circMYO1C showed a higher expression as compared to control group (Figure 1B). CircMYO1C was an exonic circRNA spliced from MYO1C gene exon9-exon8 via back splicing, thus named as circMYO1C, which was confirmed by Sanger sequencing (Figure 1C). To detect the stability of circMYO1C, PANC-1 cells were treated with RNase R (RNA synthesis inhibitor) and actinomycin D (DNA repair inhibitor). The half-life time of circMYO1C was longer than MYO1C mRNA (Figure 1D). qRT-PCR results showed that circMYO1C was more capable of resistance to RNase R digestion (Figure 1E). RNA fluorescence in situ hybridization (RNA-FISH) displayed that the circMYO1C distributed in the cytoplasm of PDAC cells (Figure 1F). In the clinical specimens of PDAC patients, quantitative analysis found that the expression of circMYO1C up-regulated in PDAC as compared to normal controls (Figure 1G). Taken together, these findings indicated that circMYO1C was a METTL3-induced circRNA and up-regulated in PDAC.
3.2. circMYO1C promoted the proliferation and migration of PDAC cells
In PDAC cells, we found that the circMYO1C expression was up-regulated as compared to normal cells (Figure 2A). To investigate the functions of circMYO1C on PDAC cells, the enforced overexpression and silencing of circMYO1C were respectively transfected into Capan-2 cells (vector, circMYO1C overexpression) and PANC-1 cells (sh-NC, sh-circMYO1C) (Figure 2B). CCK-8 proliferation assay indicated that circMYO1C overexpression promoted the proliferative ability of PDAC cells and circMYO1C silencing repressed the proliferation (Figure 2C). Migration assay indicated that circMYO1C overexpression promoted the migrative ability of PDAC cells and circMYO1C silencing repressed the migration (Figure 2D). Wound healing assay unveiled that enhanced ircMYO1C accelerated the migrative ability, while the knockdown of circMYO1C inhibited the migration (Figure 2E). Ethynyl-2-deoxyuridine (EdU) incorporation assay illustrated that circMYO1C overexpression promoted the DNA synthesis, while the knockdown of circMYO1C repressed the DNA synthesis (Figure 2F). Overall, these data demonstrated that circMYO1C promoted the proliferation and migration of PDAC cells.
3.3. METTL3 induced the expression of circMYO1C via m6A-modified manner
Latest research shows that METTL3 could regulate the biogenesis of circRNAs, thus we investigate the interaction within METTL3 and circRNAs. Given that circMYO1C was up-regulated in the sequencing upon METTL3 overexpression, we proposed a hypothesis that METTL3 induced the circularization of circMYO1C. Firstly, the up-regulated or down-regulated METTL3 construction was performed, whose transfection efficiency was identified by western blot (Figure 3A). Then, RT-qPCR analysis fund that circMYO1C level was upregulated upon METTL3 overexpression transfection, while circMYO1C level was repressed upon METTL3 silencing (Figure 3B). MeRIP-qPCR analysis found that the m6A-modified enrichment was higher in the PDAC cells (Capan-2, PANC-1) (Figure 3C). RNA pull-down following western blot assays further verified that METTL3 could interact with circMYO1C in Capan-2 cells (Figure 3D). Overall, these findings unveiled METTL3 induced the expression of circMYO1C via m6A-modified manner.
3.4. circMYO1C promoted the VEGFA mRNA stability
MeRIP-Seq revealed that several candidate genes (MYC, VEGFA, HOXA10, Notch1) demonstrated the m6A modification in their genomic location (Figure 4A). Then, RT-qPCR analysis revealed that circMYO1C overexpression significantly up-regulated the VEGFA mRNA level, while circMYO1C knockdown reduced the expression of VEGFA mRNA (Figure 4B, 4C). Meanwhile, the other candidates showed no significant change. Moreover, RNA stability assay using Act D administration showed that circMYO1C overexpression remarkably increased the VEGFA mRNA stability and circMYO1C knockdown repressed the VEGFA mRNA stability (Figure 4D). Taken together, these findings found that VEGFA exhibited the m6A modification and circMYO1C promoted the VEGFA mRNA stability.
3.5. circMYO1C interacted with VEGFA through m6A reader IGF2BP2
Previous research has reported that circRNA could regulate its target mRNA stability, which is mediated by m6A reader IGF2BP216. For the further mechanism by which circMYO1C regulates the stability of VEGFA mRNA, our research put forward a hypothesis that circMYO1C might interact with VEGFA through m6A reader IGF2BP2. Analytical investigation revealed that IGF2BP2 shared the potential m6A modified sites with both circMYO1C junction sites and VEGFA 3’-UTR (Figure 5A). RNA binding protein immunoprecipitation (RIP) analysis proved that, comparing with the control IgG, circMYO1C expression was enriched in the anti-IGF2BP2 antibody precipitation (Figure 5B). RNA pull-down assay using circMYO1C probe confirmed that circMYO1C specifically combined with VEGFA (Figure 5C). As we known, IGF2BP2 possesses 2 RNA-recognition-motif (RRM) domains and 4 K homology (KH) domains17, 18. Thus, the next work for our team was to explore which domain might combine with circMYO1C. FLAG tagged full-length and truncated IGF2BP3 mutants were constructed (Figure 5D). RIP analysis proved KH3-KH4 domains of IGF2BP2 specifically interacted with circMYO1C, which was required for its interaction with circMYO1C and VEGFA mRNA (Figure 5E). RIP analysis demonstrated that circMYO1C overexpression enhanced the IGF2BP2-VEGFA interaction, while circMYO1C knockdown reduced the protein-RNA interaction (Figure 5F). By performing RNA fluorescence in situ hybridization (RNA-FISH) assays, we confirmed the colocalization of endogenously expressed circMYO1C and VEGFA in the cytoplasm (Figure 5G). Overall, these findings unveiled that circMYO1C interacted with VEGFA through m6A reader IGF2BP2.
3.6. circMYO1C knockdown repressed the tumor growth in vivo
To investigate the role of circMYO1C in vivo, the xenograft in vivo mice assay was performed. Results indicated that circMYO1C knockdown reduced the tumor weight (Figure 6A) and volume (Figure 6B). Immumohistochemical staining (IHC) showed that the VEGFA protein was decreased upon circMYO1C knockdown (Figure 6C). Bioluminescence in vivo imaging showed that circMYO1C knockdown repressed the tumor metastasis (Figure 6D). Overall, these finding illustrated that circMYO1C knockdown repressed the tumor growth in vivo.