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Objective. To explore the substrate of F-box only protein 22 (FBXO22) and its mechanism, and further study the role of FBXO22 in cancer cell apoptosis. Methods. The interaction protein of FBXO22, namely apoptosis-inducing factor (AIF), was searched and identified by immunoprecipitation (IP) -coupled liquid chromatograph mass spectrometer/mass spectrometer (LC-MS/MS) technology. The effect of FBXO22 on ubiquitination of AIF was detected by ubiquitin modification assay under denatured conditions. Short hairpin RNA (shRNA) knockdown FBXO22 and overexpression FBXO22 were used to detect the expression of AIF. The effect of FBXO22 knockdown or overexpression on apoptosis of colon cancer cells was detected by flow cytometry. Results. IP-coupled LC-MS/MS technology showed that FBXO22 could interact with AIF, and ubiquitin modification assay under denaturing conditions showed that FBXO22 could mediate ubiquitination of AIF. Knockdown of FBXO22 could up-regulate the level of AIF, while overexpression of FBXO22 could down-regulate the level of AIF. The results of flow cytometry showed that knockdown of FBXO22 could promote the apoptosis of colon cancer cells induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while overexpression of FBXO22 could inhibit the apoptosis of colon cancer cells induced by MNNG. Conclusion. FBXO22 regulates AIF-mediated apoptosis of colon cancer cells by ubiquitination and degradation of AIF. [ABSTRACT FROM AUTHOR]