Effect of p53/Numb protein functional complex on apoptosis of colon cancer cells
Objective To explore the effect of p53 protein on the expression of Numb protein in colorectal cancer and its action mechanism,observe the interaction between the two on the ubiquitination degradation of Numb protein and the impact on apoptosis of colon cancer cells.Methods Human colon cancer cell lines HCT116(+)and HCT116(-)[p53 wild-type and p53 deletion type,American Center for the Collection of Typical Strains(ATCC)source]were used as research subjects.Gene overexpression(Transfer)and RNA interference(RNAi)were used respectively,and the cells were treated or not treated with proteasome inhibitor MG132 for 12 h.The effect of p53 protein on the expression level of Numb pro-tein and the stability of its ubiquitin proteasome pathway was detected using Western blotting,and the effect of p53/Numb on apoptosis of colon cancer cells was detected using flow cytometry.Analysis of variance(ANOVA)was used for inter group comparison,and independent sample t-test was used to compare protein expression differences.Statistical methods included independent sample t-test,independent sample t-test,and one-way analysis of variance.Results The protein level of Numb increased in a dose-dependent man-ner as the protein expression level of p53 gradually increased.In the blank group and p53 transfection group(2 μg and 4 μg),the ratio of Numb protein expression levels was 0.632(0.633±0.008)and 0.137(0.136±0.003),respectively(t=5.332,P＜0.01),while the mRNA level of Numb showed no signifiicant change with the increase of p53 mRNA expression level[0.892(0.890±0.003)vs.0.826(0.825±0.006),t=1.232,P＞0.05].After 48 h of p53 RNA interference or no interference,the pro-tein level of Numb was lower than that in the control group[0.167(0.163±0.001)and 0.536(0.532± 0.005),t=3.374,P＜0.05],and the mRNA level of Numb showed no significant change with the de-crease of p53 mRNA expression level.After p53 RNA interference,the expression level of Numb protein decreased,while the proteasome inhibitor MG132 could make the protein level of Numb tend to be consist-ent[0.753(0.750±0.009)vs.0.742(0.744±0.006),t=1.532,P＞0.05].When MG132 was not present,the level of Numb protein in the presence of p53 was higher than that in the absence of p53,but the level of Numb protein was still lower than that in the presence of MG132.The confocal results showed that in the absence of p53,Numb was mainly localized in the cell membrane and cytoplasm,exhibiting a diffuse distribution.When p53 was present,Numb was localized in the cytoplasm,exhibiting an aggregated distribution and increased expression level.However,some p53 was also localized in the cytoplasm,indi-cating that Numb and some p53 were co localized in the cytoplasm.In HCT116(p53+),the effect of p53 RNA interference was opposite.In HCT116(+),after being transferred to Numb for 48 h,the number of apoptotic cells significantly increased(13.7％vs.48.3％,t=4.523,P＜0.01),and there was a syner-gistic effect with DAPT in the same direction(t=4.523,P＜0.01).In HCT116(-),the number of ap-optotic cells also increased,but the effect was slightly weaker than the former,while the corresponding blank control group and empty group showed no significant changes.Conclusion p53 in colon cancer cells affects its protein stability through the ubiquitin proteasome pathway by binding to Numb protein,and may affect colon cancer cell apoptosis through the Numb/Notch pathway.
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