Figure 3.

Gut microbiota regulates the expression of circRNAs. A-B, The circulating circRNA expression profile from GF/SPF and GF mice was compared using a high-throughput circRNA microarray. C, Interaction predictive analysis of target microRNAs and target circRNAs, which are regulated by gut microbiota. D-E, The differentially expressed circRNAs were identified, and qRT-PCR was used to verify a subset of the differentially expressed circulating circRNAs. F-G, Circulating mmu_circ_0000730 were detected in GF, GF/Bifido groups by RT-qPCR, and was further verified by qRT-PCR between GF and GF/Bifido group in tumor tissue. Statistical analysis: t test. H-I, Competitive regulation of mmu_circ_0000730 and mmu-miR-466i-3p/mmu-miR-466 f-3p. J-K, Dual-luciferase reporter assay:interaction analysis of mmu_circ_0000730 and mmu-miR-7 f-1-3p (j), mmu_circ_0000730 and mmu-miR-466i-3p (k). L, RNA pulldown by designed nucleic acid probes for target circRNAs to detect Ago2 protein in the RISC protein complex followed with western blot. M-N, RIP experiment with Ago2 antibody to check the target circRNAs including mmu_circ_0000730 (m) or miRNAs (n) by qRT-PCR. Statistical analysis: two-way ANOVA (D,E,H,I,J,K,M & N). Data are shown as mean ± SEM from three experiments performed in triplicates. *P < .05; **P < .01; ***P < .001; ns, not significant.