Fig. 8

Mir-223-3p targets E3 ligase CBLB to regulate ubiquitination of cMYC. A Venn diagram indicated that the overlap of downstream genes of miR-223-3p predicted by miRDB, miRWalk and TargetScan database. B-C qRT-PCR and western blot showed that the expression of CBLB was conspicuously decreased or increased upon miR-223-3p overexpression or knockdown respectively in LoVo cells, while the difference of SIAH1 and FBXW7 was not significant. D A miR-223-3p binding site on the 3'UTR of the CBLB mRNA was predicted by TargetScan database. E The binding of miR-223-3p on CBLB 3’UTR was evaluated by dual luciferase reporter assay.293 T cells were transfected with reporter plasmid containing wild type CBLB 3'UTR(WT) or mutant type (MUT) respectively, followed by transfection with mir-223-3p mimic (miR-mimic) or negative control (miR-NC). F Western blot showed that the expression of proliferation and migration markers and cMYC was significantly decreased or increased in colon cancer cells upon CBLB overexpression or knockdown in colon cancer cells. G Colon cancer cells were overexpressed CBLB followed by treatment with cycloheximide (CHX) for the indicated times. The intensity of cMYC expression at each time point was quantified by densitometry and plotted against time. H Ubiqutin assays of colon cancer cells transfected Sh-CBLB followed by treatment with MG132. I CO-IP and Western blot showed that endogenous cMYC and CBLB bind to each other. J Immunofluorescence images showed colocalization of CBLB and cMYC in colon cancer cells. All data were revealed as mean ± standard deviation (SD) for no less than three independent experiments. Significant P values showed as ^***P < 0.001.^**P < 0.01. ns means the difference was not significant