14-3-3σ Regulates HIF1α Protein Expression In Colon Cancer Via Ubiquitin Proteasome Pathway Under Hypoxic Conditions

Objective:Hypoxia-inducible factor 1 alpha(HIF1α)is a tumor-promoting factor.In this study,we explored the regulation of 14-3-3σ on HIFla and its underlying mechnism with hypoxic conditions in vitro.Methods:1.Analyze the expression of 14-3-3σ in colon cancer and its effect on the prognosis of colon cancer patients by bioinformatics method,and confirm the expression pattern of 14-3-3σ in multiple colon cancer cell lines by Western blotting.2.Screen the colon cancer cell lines which express HIF1α consistently under hypoxic condition by Western blotting.3.14-3-3σ overexpression plasmids(pCMV5-FLAG-14-3-σ)were transfected into the selected colon cancer cell lines DLD1 and HCT15.Detect the HIF1αexpression after hypoxia stimulation at mRNA and protein levels through qRT-PCR and Western blotting.14-3-3σ 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 HIF1α protein in 14-3-3σ knockdown DLD1 cell lines was reversely verified.4.Confirmed the effect of 14-3-3σ on the HIFla stability in 14-3-3σ overexpressed DLD1 cell under hypoxic condition by CHX assay,and the ubiquitination level of HIF1αwas detected by in vitro ubiquitination assay.Results:1.The expression of 14-3-3σ is generally low in colon cancer,which is associated with poor prognosis in patients with CRC.2.The expression of HIF1α in colon cancer cells DLD1 and HCT15 were gradually increased over time under 1%O2 hypoxia.3.Overexpression of 14-3-3σ effectively inhibited the accumulation of HIF1α protein in 14-3-3σ overexpressed cell lines DLD1 and HCT15.Conversely,the accumulation of HIF1α in 14-3-3σ knockdown DLD1 cell lines were significantly increased compared to the control group.4.Overexpression of 14-3-3σ could affect the mRNA level of HIFla in DLD1 cell line,but its expression was inconsistent with the protein.5.Overexpression of 14-3-3σ significantly reduced the stability of HIFla in DLD1 cells under the condition of 1%O2 hypoxia.One of the mechanisms that 14-3-3σinhibited the expression of HIF1α may through increasing the ubiquitination of HIF1α,HIF1α was therefore degraded via ubiquitin-proteasome pathway.Conclusion:This study revealed that 14-3-3σ inhibits HIF1α expression in colon cancer under hypoxic conditions,and that the ubiquitin-proteasome pathway is a potential regulatory mechanism.