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Figure 1. Identification of circRNAs associated with responsiveness or resistance to PD-1 antibody therapy of CRC. (A) Heat map
showing the differentially expressed circRNAs in CRC samples responsive (n = 13) or resistant (n = 19) to PD-1 antibody treatment
analyzed by circRNA microarray; (B) Volcano plot showing circRNAs differentially expressed in tissues responsive or resistant to PD-1
antibody treatment. Cut off is Log 2 (Fold Change) > 1.5, P < 0.05; (C) Procedure to select the key circRNA involved in CRC progression
and tumor immune evasion; (D and E) Relative expression of circNCOA3 in primary CRC tissues (D) and in the serum (E) of patients
responsive (n = 22) and resistant (n = 33) to PD-1 antibody treatment in a separate cohort (P < 0.001); (F and G) Kaplan-Meier analysis
of the association of circNCOA3 expression level with OS (F) and PFS (G) in CRC patients treated with PD-1 antibody; (H)
Representative pictures of circNCOA3 expression (detected by ISH) and PD-L1 expression (detected by IHC) in CRC tissues; (I)
Expression of circNCOA3 in the PD-L1 high and PD-L1 low expression groups. circRNAs: Circular RNAs; PD-1: programmed cell death 1;
CRC: colorectal cancer; OS: overall survival; PFS: progression-free survival; IHC: immunohistochemistry.

shRNA plasmids targeting the unique back-splice junction of circNCOA3, the expression of circNCOA3
was significantly knocked down [Figure 3B]. The CCK-8 assay showed that the cell viability was markedly
reduced in CRC cells after the knockdown of circNCOA3 [Figure 3C and D]. Colony formation assay
revealed that cell proliferation was significantly suppressed in CRC cells after circNCOA3 knockdown as