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Chen et al. Cancer Drug Resist 2024;7:9 https://dx.doi.org/10.20517/cdr.2023.151 Page 7 of 15

Figure 3. circNCOA3 promotes CRC progression. (A) Levels of circNCOA3 were measured by RT-qPCR in normal colon cells
(NCM460) and various CRC cell lines; (B) Levels of circNCOA3 in CSC cell lines HCT116 and SW620 transfected with shRNAs; (C and
D) Proliferation assays in cell lines HCT116 (C) and SW620 (D) transfected with sh-circNCOA3; (E) Colony formation assays of the
CRC cell lines HCT116 and SW620 transfected with sh-circNCOA3; (F) Transwell assay of the CRC cell lines HCT116 and SW620
transfected with sh-circNCOA3; (G) Tumor growth in C57BL/6 mice implanted with MC38-sh-NC or MC38-sh-circNCOA3 cells; (H)
Tumor growth in BALB/c nude mice implanted with MC38-sh-NC or MC38-sh-circNCOA3 cells; (I) The OS in different groups analyzed
using the Kaplan-Meier method. CRC: Colorectal cancer; RT-qPCR: real-time polymerase chain reaction; OS: overall survival.

indicated by reduced colony formation numbers [Figure 3E]. In Transwell experiments, the cell invasive
ability of CRC cells was significantly suppressed after circNCOA3 knockdown [Figure 3F]. To investigate
the in vivo role of circNCOA3, we first generated MC38-sh-circNCOA3 cell lines by transfecting
circNCOA3-targeting shRNA vectors into the MC38 cell line. MC38-sh-circNCOA3 cells and control cells
were injected subcutaneously into immunocompetent mice (C57BL/6 mice) or immunodeficient mice
(BALB/c nude mice). The results showed that tumor growth was significantly inhibited in
immunocompetent mice after knockdown of circNOCA3 [Figure 3G]. However, no obvious difference in
tumor growth was observed in immunodeficient mice after circNOCA3 knockdown [Figure 3H]. Moreover,
the survival time was significantly longer after circNOCA3 knockdown in immunocompetent mice
[Figure 3I]. IHC experiments showed that MC38-sh-circNCOA3-derived tumors presented a high amount