Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Nov 29;13(11):1008.
doi: 10.1038/s41419-022-05451-y.

m6A-modified circFNDC3B inhibits colorectal cancer stemness and metastasis via RNF41-dependent ASB6 degradation

Wei Zeng #  1   2 , Jin-Feng Zhu #  3 , Jian Guo  1 , Gen-Jie Huang  1 , Li-Sha Ai  4 , Yu Zeng  1 , Wang-Jun Liao  5
Affiliations

m6A-modified circFNDC3B inhibits colorectal cancer stemness and metastasis via RNF41-dependent ASB6 degradation

Wei Zeng et al. Cell Death Dis. .

Abstract

Colorectal cancer (CRC) is the third most frequently diagnosed cancer with unfavorable clinical outcomes worldwide. circFNDC3B plays as a tumor suppressor in CRC, however, the mechanism of circFNDC3B in CRC remains ambiguous. The stem-like properties of CRC cells were detected by the evaluation of stemness markers, sphere formation assay and flow cytometry. qRT-PCR, FISH, IHC, and western blotting assessed the expression and localization of circFNDC3B, RNF41, ASB6, and stemness markers in CRC. The metastatic capabilities of CRC cells were examined by wound healing and Transwell assays, as well as in vivo liver metastasis model. Bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assay and co-IP were used to detect the associations among circFNDC3B, FXR2, RNF41, and ASB6. Downregulated circFNDC3B was associated with unfavorite survival in CRC patients, and circFNDC3B overexpression suppressed CRC stemness and metastasis. Mechanistically, studies revealed that YTHDC1 facilitated cytoplasmic translocation of m6A-modified circFNDC3B, and circFNDC3B enhanced RNF41 mRNA stability and expression via binding to FXR2. circFNDC3B promoted ASB6 degradation through RNF41-mediated ubiquitination. Functional studies showed that silencing of RNF41 counteracted circFNDC3B-suppressed CRC stemness and metastasis, and ASB6 overexpression reversed circFNDC3B- or RNF41-mediated regulation of CRC stemness and metastasis. Elevated ASB6 was positively correlated with unfavorite survival in CRC patients. In vivo experiments further showed that circFNDC3B or RNF41 overexpression repressed tumor growth, stemness and liver metastasis via modulating ASB6. Taken together, m6A-modified circFNDC3B inhibited CRC stemness and metastasis via RNF41-dependent ASB6 degradation. These findings provide novel insights and important clues for targeted therapeutic strategies of CRC.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. circFNDC3B is downregulated in CRC tissues and cells, and low circFNDC3B expression is associated with unfavorable overall survival in CRC patients.
A Schematic drawing illustrated the formation of circFNDC3B. B, C The expression level of circFNDC3B was detected by qRT-PCR in LoVo and HCT116 cells treated with Actinomycin D or RNase R. D The circFNDC3B level in CRC tissues was detected by qRT-PCR. E The correlation between circFNDC3B level and OS of CRC patients was analyzed by Kaplan–Meier method. F The circFNDC3B level in CRC cells was detected by qRT-PCR. G The circFNDC3B level in LoVo and HCT116 cells was detected by subcellular fractionation. H The subcellular localization of circFNDC3B was detected by RNA FISH in LoVo and HCT116 cells. Scale bar, 10 μm; Green, circFNDC3B; Blue, DAPI. **P < 0.01, ***P < 0.001.
Fig. 2
Fig. 2. circFNDC3B suppresses CRC stemness and metastasis.
A The circFNDC3B level in transfected CRC cells was detected by qRT-PCR. B Representative sphere images of transfected CRC cells. C CD133+ cells were analyzed by flow cytometry with quantitative analysis in CRC cells transfected with circFNDC3B overexpression vector or sh-circFNDC3B. D Cell migration was monitored by wound healing assay with quantitative analysis in CRC cells transfected with circFNDC3B overexpression vector or sh-circFNDC3B. E Cell invasion was measured by Transwell invasion assay with quantitative analysis in CRC cells transfected with circFNDC3B overexpression vector or sh-circFNDC3B. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3
Fig. 3. YTHDC1 promotes cytoplasmic translocation of m6A modified circFNDC3B.
A m6A modification of circFNDC3B was detected by MeRIP-qPCR. B The interaction between biotinylated circFNDC3B and YTHDC1 was verified by RNA pull-down assay. Random probe acted as a negative control. C The interaction between circFNDC3B and YTHDC1 was detected by RIP assay. Normal IgG served as a negative control. D The circFNDC3B level in CRC cells was detected by subcellular fractionation and qRT-PCR. E The subcellular localization of circFNDC3B was detected by RNA FISH. Scale bar, 10 μm; Green, circFNDC3B; Blue, DAPI. **P < 0.01, ***P < 0.001.
Fig. 4
Fig. 4. circFNDC3B increases RNF41 mRNA stability and expression via binding to FXR2.
A The RNF41 level in CRC tissues was detected by qRT-PCR. B The correlation between circFNDC3B and RNF41 level in CRC tissues was analyzed by spearman correlation analysis. C The interaction between biotinylated circFNDC3B and FXR2 was detected by RNA pull-down assay. Random probe acted as a negative control. D The interaction between circFNDC3B and FXR2 was detected by RIP assay. Normal IgG served as a negative control. E The co-localization of circFNDC3B and FXR2 was detected by RNA FISH and immunofluorescence (IF). Scale bar, 10 μm; Green, circFNDC3B; Red, FXR2; Blue, DAPI. F, G The interaction between RNF41 and FXR2 was detected by RIP assay. Normal IgG served as a negative control. H The mRNA stability of RNF41 was determined by RNA stability assay. I, J The mRNA and protein levels of RNF41 in transfected CRC cells were detected by qRT-PCR and Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5
Fig. 5. Knockdown of RNF41 counteracts circFNDC3B-suppressed CRC stemness and metastasis.
A–E The expression of stemness markers were detected by qRT-PCR and Western blotting. F Representative sphere images of transfected CRC cells with quantitative analysis. G CD133+ cells were analyzed by flow cytometry with quantitative analysis in transfected CRC cells. H Cell migration was monitored by wound healing assay with quantitative analysis in transfected CRC cells. I Cell invasion was measured by Transwell invasion assay with quantitative analysis. *P < 0.05, **P < 0.01.
Fig. 6
Fig. 6. circFNDC3B promotes ASB6 degradation through RNF41-mediated ubiquitination.
A The protein level of ASB6 was determined by western blotting with quantitative analysis. B The ubiquitination of ASB6 was assessed by co-IP in transfected CRC cells. C The interaction between ASB6 and RNF41 was detected by Co-IP. Normal IgG served as a negative control. D The protein levels of RNF41 and ASB6 were determined by western blotting with quantitative analysis in transfected CRC cells. E The protein level of ASB6 was determined by western blotting with quantitative analysis in transfected CRC cells. F The interaction between ASB6 and RNF41 in circFNDC3B-overexpressing cells was detected by Co-IP. *P < 0.05, **P < 0.01.
Fig. 7
Fig. 7. ASB6 is elevated in CRC and associated with poor OS in CRC patients.
A, B Differential expression of ASB6 and survival analysis in CRC based on UALCAN database. C The mRNA level of ASB6 in CRC tissues was detected by qRT-PCR. D, E The correlation among circFNDC3B, RNF41 and ASB6 in CRC tissues was analyzed by spearman correlation analysis. F The protein level of ASB6 in CRC tissues was detected by western blotting. G The correlation between ASB6 level and OS of CRC patients was analyzed by Kaplan–Meier method. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 8
Fig. 8. Overexpression of ASB6 reverses circFNDC3B or RNF41-mediated regulation of CRC stemness and metastasis.
A–E The expression of stemness markers were detected by qRT-PCR and western blotting. F Representative sphere images of transfected CRC cells with quantitative analysis. G CD133+ cells were analyzed by flow cytometry with quantitative analysis in transfected CRC cells. H Cell migration was monitored by wound healing assay with quantitative analysis in transfected CRC cells. I Cell invasion was measured by Transwell invasion assay with quantitative analysis. *P < 0.05, **P < 0.01.
Fig. 9
Fig. 9. Overexpression of circFNDC3B or RNF41 suppresses tumor growth, stemness and liver metastasis via modulating ASB6 in vivo.
A Photographs of xenograft tumors. B Quantitative analysis of tumor volume. C Quantitative analysis of tumor weight. D–H The mRNA levels of RNF41, OCT4, Nanog, SOX2, and CD133 in xenograft tumors were detected by qRT-PCR. I The protein levels of ASB6, OCT4, Nanog, SOX2, and CD133 were detected by western blotting with quantitative analysis. J The immunoreactivities of Ki-67, CD133, and ASB6 in xenograft tumors were detected by IHC analysis. K Representative photographs of liver tissues derived from in vivo liver metastasis mice model. L The histological changes of liver tissues were detected by H&E staining. *P < 0.05, **P < 0.01, ***P < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7–33. doi: 10.3322/caac.21708. - DOI - PubMed
    1. Siegel RL, Miller KD, Goding Sauer A, Fedewa SA, Butterly LF, Anderson JC, et al. Colorectal cancer statistics, 2020. CA Cancer J Clin. 2020;70:145–64. doi: 10.3322/caac.21601. - DOI - PubMed
    1. Dienstmann R, Vermeulen L, Guinney J, Kopetz S, Tejpar S, Tabernero J. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat Rev Cancer. 2017;17:268. doi: 10.1038/nrc.2017.24. - DOI - PubMed
    1. Coker H, Wei G, Brockdorff N. m6A modification of non-coding RNA and the control of mammalian gene expression. Biochim Biophys Acta Gene Regul Mech. 2019;1862:310–8. doi: 10.1016/j.bbagrm.2018.12.002. - DOI - PubMed
    1. Yang X, Hu X, Liu J, Wang R, Zhang C, Han F, et al. N6-methyladenine modification in noncoding RNAs and its function in cancer. Biomark Res. 2020;8:61. doi: 10.1186/s40364-020-00244-x. - DOI - PMC - PubMed

Publication types