The Molecular Mechanism Of Ubiquitin E3 Ligase TRIM21 Regulating MICALL2 In Colorectal Cancer Cells

ObjectiveTo explore the interaction and interaction mode of TRIM21 and MICALL2,clarify the molecular mechanism of TRIM21 regulating MICALL2,and whether TRIM21 relies on MICALL2 to regulate the proliferation and migration of colorectal cancer cells.Methods1.In this study,MICALL2 was used for immunoprecipitation assays and mass spectrometry analysis to find the potential interacting protein TRIM21.The interaction between them was proved by immunoprecipitation assays and immunofluorescence techniques.2.Plasmids with different truncations of MICALL2 and TRIM21 were constructed,and their interaction domains were detected by immunoprecipitation test.3.Overexpression or silencing of TRIM21 in colorectal cancer cell lines,Western blot was used to detect the change level of MICALL2.4.Overexpression of TRIM21 in colorectal cell lines was detected by immunoprecipitation to detect the ubiquitination level of MICALL2,and then the change level of MICALL2 was detected by the intervention of proteasome-specific inhibitors MG132 and protein synthesis inhibitor CHX(actinomycin).5.Obtain data about colorectal cancer from The Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA).Evaluate the expression level of TRIM21 m RNA in tumor tissue and analyzethe correlation between MICALL2 m RNA and TRIM21 m RNA using the GEPIA tool.Then the expression level of TRIM21 and MICALL2 in colorectal cancer tissues and their correlation were detected by immunohistochemistry.6.To determine whether TRIM21 depends on MICALL2 to regulate the proliferation and migration of colorectal cancer cells by MTT and cell scratch test.Results1.The results of immunoprecipitation and immunofluorescence mutually verified the interaction between MICALL2 and TRIM21 in colorectal cancer cells.The results of immunoprecipitation between the domain fragments showed that the C-terminal PRY-SPRY domain of TRIM21 is required to interact with MICALL2,whereas the b MERB domain is essential for MICALL2 to bind TRIM21.2.Silencing TRIM21 increased the protein level of MICALL2;Overexpression of TRIM21 significantly reduced the protein level of MICALL2 and increased the ubiquitination of MICALL2.3.The degradation of MICALL2 protein mediated by overexpression of TRIM21 can be antagonized by treatment with MG132.In addition,Overexpression of TRIM21 significantly shortened the half-life of MICALL2.4.According to the analysis of CRC data in the TCGA database,there was no discernable relationship between TRIM21 and MICALL2 at the transcriptional level.Furthermore,the tissues with lower expression of TRIM21 have strong staining of MICALL2,in contrast,the samples with higher expression of TRIM21 displayed low levels of MICALL2 expression.5.MTT tests and wounding healing assay results showed that the decreased proliferation and migration ability of colorectal cancer cells silencing MICALL2 could be restored by silencing TRIM21.ConclusionsTRIM21 can interact with MICALL2,and TRIM21 ubiquitin modifies MICALL2 and down-regulates MICALL2 protein level through the proteasome pathway.In addition,TRIM21 relies on MICALL2 to regulate the proliferation and migration of colorectal cancer cells.