14-3-3? Regulates HIF1? Protein Expression In Colon Cancer Via Ubiquitin Proteasome Pathway Under Hypoxic Conditions

Objective:Hypoxia-inducible factor 1 alpha(HIF1?)is a tumor-promoting factor.In this study,we explored the regulation of 14-3-3? on HIFla and its underlying mechnism with hypoxic conditions in vitro.Methods:1.Analyze the expression of 14-3-3? in colon cancer and its effect on the prognosis of colon cancer patients by bioinformatics method,and confirm the expression pattern of 14-3-3? in multiple colon cancer cell lines by Western blotting.2.Screen the colon cancer cell lines which express HIF1? consistently under hypoxic condition by Western blotting.3.14-3-3? overexpression plasmids(pCMV5-FLAG-14-3-?)were transfected into the selected colon cancer cell lines DLD1 and HCT15.Detect the HIF1?expression after hypoxia stimulation at mRNA and protein levels through qRT-PCR and Western blotting.14-3-3? knockdown plasmid(pLKO.1-shSFN)was transfected into 293T cells to harvest lentiviruses,which was used to infect colon cancer cell DLD1.The regulation of HIF1? protein in 14-3-3? knockdown DLD1 cell lines was reversely verified.4.Confirmed the effect of 14-3-3? on the HIFla stability in 14-3-3? overexpressed DLD1 cell under hypoxic condition by CHX assay,and the ubiquitination level of HIF1?was detected by in vitro ubiquitination assay.Results:1.The expression of 14-3-3? is generally low in colon cancer,which is associated with poor prognosis in patients with CRC.2.The expression of HIF1? in colon cancer cells DLD1 and HCT15 were gradually increased over time under 1%O2 hypoxia.3.Overexpression of 14-3-3? effectively inhibited the accumulation of HIF1? protein in 14-3-3? overexpressed cell lines DLD1 and HCT15.Conversely,the accumulation of HIF1? in 14-3-3? knockdown DLD1 cell lines were significantly increased compared to the control group.4.Overexpression of 14-3-3? could affect the mRNA level of HIFla in DLD1 cell line,but its expression was inconsistent with the protein.5.Overexpression of 14-3-3? significantly reduced the stability of HIFla in DLD1 cells under the condition of 1%O2 hypoxia.One of the mechanisms that 14-3-3?inhibited the expression of HIF1? may through increasing the ubiquitination of HIF1?,HIF1? was therefore degraded via ubiquitin-proteasome pathway.Conclusion:This study revealed that 14-3-3? inhibits HIF1? expression in colon cancer under hypoxic conditions,and that the ubiquitin-proteasome pathway is a potential regulatory mechanism.