Molecular Mechanism Of SPOP Inhibits The Hepatocellular Carcinoma Cell Migration And Invasion

Part 1 Low SPOP expression correlates with the malignant biological behaviors in hepatocellular carcinomaObjective: SPOP plays an inhibiting role in several kinds of tumors,but its role in hepatocellular carcinoma(HCC)is to be elucidated.Therefore,the aim of our study is to observe SPOP expression and its clinical significance in HCC.Methods: We detected SPOP mRNA levels in 74 pairs of HCC samples by quantitative real-time polymerase chain reaction,and examined SPOP protein expression in another 44 paired HCC and adjacent non-tumor tissues by immunohistochemistry staining and western blotting.The correlations between SPOP and clinicopathologic features were also analyzed.Cox proportional hazard model was used to analyze the correlations between low SPOP expression and prognosis of patients.Results: 85.1%(63/74)of SPOP mRNA transcriptional levels detected by qRT-PCR were significantly lower in HCC tissues compared with paired adjacent normal tissues(P < 0.001)from 74 clinical patients.Western blottingdemonstrated that 84.1%(37/44)of SPOP protein levels were also lower in cancer tissues than those inpaired adjacent normal tissues from the same patient specimens(P< 0.01).Immunohistochemistry staining results showed that SPOP was mainly expressed in the cytoplasm and nuclei in matched adjacent normal tissues in a different series of 44 matched clinical samples,but 59.1%(26/44)of its protein levels were lower in cancer tissues(P< 0.05).The Chi Square Testwas usedto analyze the clinical significance of SPOP according to clinical features from44 HCC clinical patients.Low SPOP expression was significantly positively correlated with tumor size(P = 0.005)and metastasis(P = 0.002),and negativelywith differentiation(P = 0.008)and vascular invasion(P = 0.000).Furthermore,Kaplan-Meier analysis results exhibited that high SPOP expression was significantly associated with longer survival than low SPOP expression(P< 0.05).Cox's proportional hazard regression model evinced that low SPOP protein expression was a risk factor correlated with the prognosis of HCC patients(P = 0.009).Conclusion: SPOP is expressed in nucleus and cytoplasm of liver tissues.The low SPOP expression level in HCC is associated with poor prognosis,tumor size,differentiation,vascular invasion and metastasis.Low expression of SPOP is an important risk factor for patients.Part 2 SPOP suppresses HCC cell migration and invasion via ubiquitin-dependent proteolysis of SENP7Objective: To explore the molecular biological mechanism of SPOP involved in the migration and invasion of HCC.Methods: The method of packaging viruses with plasmids was used to construct the SPOP-overexpressing and SPOP-knockdown cell models and tranwell assays were selected to detect the effects of SPOP on migration and invasion of hepatocellular carcinoma cells.We used double immunofluorescence staining and co-immunoprecipitation to examine the combination between SPOP and SENP7.Subsequently,we investigated the downstream molecules of SPOP by quantitative real-time polymerase chain reaction and western blotting.Recovery experiments were used to confirm that the inhibition of migration and invasion in SPOP-overexpressing SMMC-7721 and Huh7 cells were strongly restored by SENP7.Then the effect of SPOP on SENP7 was observed by protein half-life and ubiquitination experiments.And the protein levels of SPOP,SENP7,and vimentin in 44 pairs of HCC tissues and matched adjacent normal tissues were obtained by immunohistochemistry assays.To examine the function of SPOP,we carried out the animal experiment.Results: We constructed the SPOP stable over expression of SMMC-7721 and Huh7 cells and SPOP knockdown BEL-7402 cells.SPOP inhibited the migration and invasion of HCC cells in vitro.Double immunofluorescence staining and co-immunoprecipitation results showed that SPOP could recognize and interact with SENP7 through the SPOP Math domain.SPOP was able to reduce the protein level of SENP7,but didn't affect the SENP7 mRNA level.SPOP could influence vimentin on both mRNA level and protein level.In vitro,SPOP significantly accelerated the ubiquitin level of SENP7 and reduced the half-life of SENP7 via the ubiquitin proteasome pathway.Recovery experiments showed that SPOP-mediated HCC cell invasion and metastasis partly via SENP7.IHC of patients' HCC tissue serial sections revealed that SPOP regulated the SENP7 and vimentin and was negatively associated with SENP7 and vimentin in clinical samples.Animal experiments results indicated that SPOP inhibited the liver and lung metastasis ability of HCC cells in vivo and simultaneously the protein level of SENP7 and Vimentin decreased significantly based on SPOP overexpression.Conclusion: SPOP plays a crucial role in suppressing HCC migration and invasion partly via the ubiquitination/degradation of SENP7.SPOP regulates the expression of Vimentin through SENP7 and there is SPOP-SENP7-Vimentin signaling pathway in HCC.