Study On The Mechanism Of SPOP Regulating The Biological Behavior Of Gastric Cancer Cells Via Targeting HIF-1α

Background and objective:Gastric cancer(GC)is one of the most frequent malignant tumors worldwide,and the third major reason of cancer related deaths.The 5-year overall survival rate is especially low in gastric carcinoma patients,the therapies for patients mainly including surgery(surgical operation or endoscopic surgery),radiotherapy and chemotherapy at present,whereas those had limited effect.Speckle-type POZ protein(SPOP)is an E3 ubiquitin ligase binding protein,whose mutation site is mainly located in the MATH(Meprin and TRAF-C Homology)domain,that affects substrate binding.Studies have found that SPOP could inhibit the proliferation,migration and invasion of tumors cells by ubiquitinating and degrading protein levels.Hypoxia results in the accumulation of hypoxic-inducing factors,which regulate the transcription level of various genes in cancers.Besides,Hypoxia disrupts the ubiquitination and degradation of HIF-1α,causing HIF-1α accumulates in the cytoplasm,and then alter the biological behavior of tumor cells through activating the related target genes.Researches had reported that when hypoxia occurs,HIF-1α directly induce tumor invasion and migration by interacting with Hedgehog(Hh),TGF-β and Wnt signaling pathways,leading to poor prognosis.Our previous study had found that the expression of SPOP protein was significantly reduced in gastric carcinoma tissues compared with normal gastric mucosa tissues,and SPOP protein could inhibit the proliferation,migration and invasion ability of gastric carcinoma cells through the Hh/Gli2 signaling pathway.Hence,we speculated that HIF-1α interact with SPOP protein and further influence the biological behavior of gastric carcinoma cells.Consequently,we aimed to investigate the effect and possible mechanism of SPOP/HIF-1α signal axis on the gastric cancer,and to provide new thoughts and reliable theoretical foundation for diagnosis and target therapy.Material and Methods:1.The expression of SPOP and HIF-1α protein in human gastric cancer and adjacent normal tissues were tested by immunohistochemistry.2.Kaplan-Meier curve was applied to analyze the relationship between the protein expression levels of SPOP or HIF-1α and the overall survival of gastric carcinoma patients.3.Gastric carcinoma cell model with SPOP low expression and co-cultured gastric carcinoma cell model with SPOP and/or HIF-1α low expression were established,MTT assay,plate clone formation assay and Transwell assay were applied to detect the proliferation activity,migration and invasion ability.4.The interaction between SPOP and HIF-1α in human gastric cancer cell line MKN45 were detected by co-immunoprecipitation(Co-IP)method.5.SPOP gene was knocked down in MKN45 cells and the protein expression level of downstream related genes was tested by Western blot.Results:1.The expression of SPOP and HIF-1α protein in human gastric cancer tissue and normal para-cancerous tissues and their relationship with patient prognosis(1)The protein expression level of SPOP in human gastric cancer tissues was apparently lower than corresponding para-cancerous normal tissues(P < 0.0001),and the protein expression level of HIF-1α was apparently higher in human gastric cancer tissues(P < 0.0001);(2)The overall survival rate of gastric tumor patients with higher SPOP protein expression was higher than those patients with lower expression,and the overall survival rate of gastric tumor patients with higher protein expression of HIF-1α was obviously lower than those patients with lower expression(P < 0.05).2.The effects of SPOP and HIF-1α gene on the biology of gastric carcinoma cells(1)SPOP protein was moderately expressed in gastric cancer cell line MKN45.(2)Reducing the expression of SPOP could promote the proliferation and migration of human gastric cancer cell line MKN45;(3)HIF-1α inhibitor PX-478 could inhibited the proliferation and migration of gastric cancer cells,and decreased the expression of HIF-1α can antagonize the impact of SPOP on the proliferation and migration of gastric cancer cells.3.The mechanism of SPOP regulating the biological behavior of gastric cancer cells via targeting HIF-1α(1)Co-immunoprecipitation method suggested that there was an interaction between endogenous protein SPOP and HIF-1α in gastric cancer cells,and HIF-1α was mainly bound to the N-terminal 1-189 position of SPOP amino acid(MATH domain);(2)Western blot results indicated that SPOP knock down group had lower expression of SPOP and E-cadherin protein,while had higher expression levels of HIF-1α、Vimentin protein than control group.Conclusion:1.The protein expression of SPOP and HIF-1α in gastric cancer tissues reflected an opposite tendency,and the overall survival was lower in patients with lower protein expression of SPOP or higher protein expression of HIF-1α;2.Reducing the expression of SPOP could promote the proliferation and migration of human gastric cancer cells,meanwhile,decreasing the expression of HIF-1α can antagonized the impact of SPOP on the proliferation and migration of gastric cancer cells;3.There was an interaction between SPOP and HIF-1α protein in gastric cancer cells.4.SPOP might affect the biological behavior of gastric cancer cells by targeting HIF-1α and then regulating the EMT signaling pathway.