| Background and purpose:Hepatic carcinoma is one of the most common malignancies in the world and the third leading cause of cancer death worldwide.After radical resection,the 5-year survival rate of patients with early hepatic carcinoma can reach more than 70%.However,most patients are already in the middle and late stage when diagnosed with hepatic carcinoma,when hepatic carcinoma has metastasized,which is the main cause of unsatisfactory treatment and cancer death.Therefore,it is very important to deeply understand the underlying molecular mechanism of HCC metastasis.EMT is a process in which epithelial cells lose their cellular characteristics and acquire mesenchymal phenotypes to gain the ability to invade.Many studies have confirmed that EMT plays a key role in promoting the metastasis of hepatic carcinoma.Therefore,in-depth study of the upstream regulatory mechanism of EMT is crucial for the intervention of hepatic carcinoma metastasis.NCAPG is a protein associated with meiosis and mitosis,and many studies have confirmed that NCAPG plays a significant role in the progression of tumors.However,the role of NCAPG in hepatic carcinoma EMT remains unclear.The main purpose of this study is to explore the specific mechanism of NCAPG regulating EMT in hepatic carcinoma.Methods:1.Public databases such as TCGA,GEO and CPTAC were used to analyze the expression of NCAPG in hepatic carcinoma and paracancer tissues and the relationship between NCAPG expression and patient prognosis.Western Blot,q RT-PCR and immunohistochemical tests were used to analyze the expression of NCAPG in hepatic carcinoma and paracancular tissue specimens collected in our hospital,and survival analysis was used to explore the relationship between the expression level of NCAPG and the prognosis of patients.2.Western Blot and immunofluorescence were used to analyze the expression of EMT-related proteins in hepatocellular carcinoma cells after changing the expression of NCAPG.Transewell,scratch and in vivo experiments were used to analyze the effect of changing the expression of NCAPG on the invasion and migration of hepatocellular carcinoma cells.Western Blot and immunofluorescence assay were used to analyze the expression of EMT-related proteins in hepatocellular carcinoma cells after overexpression or knockdown of NCAPG and knockdown or overexpression of β-catenin.The changes of invasion and migration ability of hepatocellular carcinoma cells after overexpression or knockdown of NCAPG while knockdown or overexpression of β-catenin were analyzed by Transewell,scratch and in vivo experiments.3.q RT-PCR was used to analyze the expression of β-catenin after changing the expression of NCAPG.Western Blot assay was used to analyze the expression ofβ-catenin over time after the addition of MG132 in hepatoma carcinoma cells,and the expression of β-catenin after the addition of MG132 and the change of NCAPG expression in hepatoma carcinoma cells.Western Blot assay was used to analyze the change of the degradation rate of β-catenin in hepatocellular carcinoma cells after changing the expression of NCAPG and adding CHX.The CO-IP assay was used to analyze the changes in the ubiquitination level of β-catenin after MG132 was added to alter the expression of NCAPG in hepatocellular carcinoma cells.4.The binding between NCAPG and β-catenin in hepatocellular carcinoma cells was analyzed by CO-IP assay;The possible binding proteins of NCAPG in hepatocellular carcinoma cells were analyzed by CO-IP mass spectrometry.The combination of NCAPG and SIP in hepatocellular carcinoma cells was analyzed by CO-IP assay.CO-IP assay was used to analyze the combination of SIP,Siah1 and Skp1 after changing the expression of NCAPG in hepatocellular carcinoma cells.Results:1.Through the analysis of public databases such as TCGA,GEO and CPTAC,it is concluded that NCAPG is highly expressed in hepatic carcinoma tissues and is closely related to poor prognosis of patients.By Western Blot,q RT-PCR and immunohistochemical analysis of hepatic carcinoma and para-cancer tissue samples collected in our hospital,it was found that NCAPG was highly expressed in hepatic carcinoma tissues,and survival analysis results showed that high expression of NCAPG indicated poor prognosis of patients.2.Western Blot and immunofluorescence analysis showed that overexpression of NCAPG in hepatocellular carcinoma cells could promote the expression of EMT-related proteins,while knockdown of NCAPG could inhibit the expression of EMT-related proteins.Transewell,scratch and in vivo analysis showed that the invasion and migration ability of hepatocellular carcinoma cells was enhanced after NCAPG overexpression,while the invasion and migration ability of hepatocellular carcinoma cells was weakened after NCAPG knockdown.Western Blot and immunofluorescence experiments showed that the expression of EMT-related proteins was enhanced after the overexpression of NCAPG in hepatoma carcinoma cells,but the expression of EMT-related proteins was significantly recovered after the knockdown of β-catenin while the overexpression of NCAPG in hepatoma carcinoma cells.After the knockdown of NCAPG in hepatoma carcinoma cells,The expression of EMT-related proteins was decreased,but the expression of EMT-related proteins was significantly restored after the knockdown of NCAPG and the overexpression ofβ-catenin.Scratch and Transwell experiments showed that the invasion and migration ability was significantly enhanced after overexpression of NCAPG in hepatocellular carcinoma cells,but the invasion and migration ability caused by overexpression of NCAPG could be recovered after knockdown of β-catenin,and the invasion and migration ability was significantly weakened after knockdown of NCAPG in hepatocellular carcinoma cells.However,NCAPG knockdown and overexpression ofβ-catenin can restore the invasion and migration ability weakened caused by NCAPG knockdown.3.q RT-PCR showed that the m RNA expression level of β-catenin did not change significantly after changing the expression of NCAPG in hepatocellular carcinoma cells.Western Blot test showed that the expression of β-catenin increased with the extension of the treatment time of MG132 in hepatocellular carcinoma cells,and the expression of β-catenin did not change with the change of NCAPG in hepatocellular carcinoma cells after MG132 was added.Western Blot analysis showed that the degradation rate of β-catenin was slow down when CHX was added after NCAPG was overexpressed in hepatocellular carcinoma cells,while the degradation rate ofβ-catenin was accelerated when CHX was added after NCAPG was knocked down.CO-IP assay showed that the ubiquitination of β-catenin was weakened after MG132 was added to hepatocellular carcinoma cells after NCAPG was overexpressed,while the ubiquitination of β-catenin was enhanced after MG132 was added to hepatocellular carcinoma cells after NCAPG was knocked down.4.The CO-IP analysis showed that NCAPG did not bind to β-catenin;IP mass spectrum analysis showed that NCAPG might combine with SIP,and COIP experiment confirmed this result.CO-IP analysis showed that after overexpression of NCAPG in hepatocellular carcinoma cells,the binding of SIP and Siah1 and Skp1 decreased,but after knockdown of NCAPG in hepatocellular carcinoma cells,the binding of SIP and Siah1 and Skp1 increased.Conclusion:The results of this study confirm that NCAPG is highly expressed in hepatic carcinoma tissues and is closely associated with poor prognosis,and elucidates the mechanism by which NCAPG inhibits β-catenin ubiquitination through competitive binding to SIP,thus promoting EMT in hepatoma carcinoma cells. |
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