| Background and Objective:Primary hepatocellular carcinoma(HCC)is currently the sixth most common malignancy in the world.It has the highest mortality rate among cancers,accounting for approximately 1% of all cancer-related deaths worldwide.Although the diagnosis and treatment of liver cancer have made great progress in the past decades,HCC patients still have a high recurrence rate and poor prognosis,which is mainly due to the high invas iveness and metastatic ability of these cancer cells.However,the process of tumor recurrence and metastasis is very complicated.This process involves multiple genes.Through these gene transcription,translation and various modification processing into a functional protein,these proteins through mutual contact activation,etc.,affect proliferation,migration,invas ion and metastasis of tumor cell.Therefore,it is necessary to develop a more reliable biomarker for predicting recurrence and understanding the mechanism of liver cancer metastasis as soon as possible.CRP is a member of the penetrating family and was the first to be discovered as an acute inflammatory protein.Its main biological function is to regulate their elimination by recruiting complementary systems and phagocytic cells through the ability to identify host pathogens and damaged cells.In recent years,studies have found that CRP is highly expressed in a variety of tumors and is closely related to tumor progression,migration,invas ion,metastasis,and proliferation and anti-apoptosis ability.Our study found that CRP is associated with clinical diagnosis and prognosis and progression of liver cancer.It was detected by isobaric Tags For Relative and Absolute Quantitation(i TRAQ)combined with tandem mass spectrometry,enzyme-linked immunosorbent assay(Elisa)and histoimmunohistochemistry to confirm that CRP can be used as an indicator of AFP diagnosis for liver cancer,especially for AFP.negative liver cancer.At the same time,we analyzed clinical data and conducted cell experiment,found that CRP is closely related to the progress ion of liver cancer.Therefore,this study extends the previous studies to explore the role of CRP in the progression of liver cancer,providing a theoretical basis for CRP as a new therapeutic target for liver cancer.Methods:1.Effect of CRP on Migration and Invasion of Hepatoma Cells(1)Detection and Significance of CRP in Hepatocellular Carcinoma Tissues and CellsReal-time quantitative PCR was used to detect the expression of CRP mRNA in 20 pairs of primary liver cancer and relative adjacent tissues.Western blot and immunohistochemistry were used to detect the expression of protein levels in 4 pairs of hepatocellular carcinoma.Further,real-time quantitative PCR and Western blot were used to detect the changes of CRP m RNA and protein levels in hepatocellular carcinoma cells with different metastatic potentials.(2)Effect of CRP on Migration and Invasion of Hepatoma CellsAfter transfection of hepatoma cells SMMC7721 and Huh7 with CRP si RNA and CRP-specific antibodies,Transwell assay was used to detect the in vitro migration and invasion of hepatoma cells.2.Screening of CRP interacting proteins(1)Co-immunoprecipitation,i TRAQ and Mass Spectrometry Screening for CRP-interacting ProteinsCRP si RNA was used as the control group and control si RNA was used as the experimental group to treat hepatoma cells SMMC7721,CRP antibody deprecipitated protein bound to CRP.The i TRAQ technique was used to mark the protein sample and identify the target protein by mass spectrometry.(2)Bioinformatics AnalysisThree online databases(DAVID,PANTHER,and Web Gestalt)were used for GO analysis,pathway analysis,and disease-related analysis.(3)CRP interacting protein validationThe candidate proteins were verified by co-immunoprecipitation and western blot.3.Effect of EphB3 on Migration and Invasion of Hepatoma Cells(1)Detection and Significance of Eph B3 in Hepatocellular Carc inoma Tissues and CellsReal-time quantitative PCR was used to detect the expression of Eph B3 m RNA in 20 pairs of primary liver cancer and relative adjacent tissues.Western blot and immunohistochemistry were used to detect the expression of protein levels in 4 liver cancer tissues.Further,real-time quantitative PCR and Western blot were used to detect the changes of Eph B3 m RNA and protein levels in hepatocellular carcinoma cells with different metastatic potential.Confocal microscopy experiments confirmed the sublocalization of Eph B3 in hepatoma cells.(2)Effect of Eph B3 on Migration and Invasion of Hepatoma CellsAfter transfection of hepatoma cells SMMC7721 and Huh7 with Eph B3 si RNA,Transwell assay was used to detect the migration and invasion of hepatoma cells.4.CRP interacts with Eph B3 to regulate the MAPK/ERK pathway(1)Interaction between CRP and Eph B3Co-immunoprecipitation experiments using CRP and Eph B3 as bait proteins were performed,and the interaction between CRP and Eph B3 was verified by western blot.(2)Relationship between Eph B3 and MAPK/ERKCo-immunoprecipitation and Western blotting verified the relat ionship between Eph B3 and MAPK/ERK,confocal assay verified the co-expression region of Eph B3 with MAPK and ERK.After transfection of hepatoma cells with Eph B3 si RNA,Western blot was used to detect the phosphorylation of MAPK/ERK.(3)Effect of CRP on MAPK/ERK Pathway,the expression of HIF1α and MMP-9The liver cancer cells were treated with CRP antibody and/or CRP recombinant protein respectively,Western blot was used to detect the changes of MAPK/ERK pathway,HIF1α and MMP9.5.Statistical methodSPSSStatistics 17.0 software performs statistical analysis.The experimental data are expressed as mean ± standard deviation(x ± s).The t test was used for comparison between the two groups.P <0.05 was considered statistically significant.Result:1.Effect of CRP on Migration and Invasion of Hepatoma Cells(1)Detection and Significance of CRP in HCC Tissues and CellsRT-PCR and Western blot were used to detect the expression of CRP in liver cancer and cell tissues.The results showed that CRP was highly expressed in hepatocellular carcinoma cells,and CRP was related to the metastatic potential of hepatoma cells.(2)Effect of CRP on Migration and Invasion of Hepatoma CellsAfter transfection of hepatoma cells with CRP si RNA or CRP Ab,Transwell assay results indicate that inhibition of CRP levels in hepatoma cells can reduce the ability of migration and invasion of hepatoma cells.2.Screening of CRP interacting proteins(1)Co-immunoprecipitation,i TRAQ and Mass Spectrometry Screening for CRP-interacting ProteinsThrough co-immunoprecipitation,i TRAQ and mass spectrometry experiments,a total of 52 proteins that may interact with CRP were identified.(2)Bioinformatics AnalysisThe GO analys is of these proteins by DAVID,PANTHER and Web Gestalt showed that these proteins are mainly located at cell junctions,cellular components,extracellular matrix,macromolecular complexes,and membranes;the main biological processes involved are cell adhes ion and regulation,trans lation,cytoskeletal organization,angiogenes is,etc.;major molecular biological functions are protein binding,cell adhes ion,cytoskeletal composition,activation of small molecules,modification,etc.The KEGG pathway analysis showed that these proteins were involved in the TRAIL signaling pathway,PI3K/AKT pathway,S1P1 pathway,membrane receptor pathway,and VEGF pathway.Disease correlation analys is showed that these proteins are mainly related to tumors,stress,chronic diseases,metabolism and metastasis.3.Effect of Eph B3 on Migration and Invasion of Hepatoma Cells(1)Detection and Significance of EphB3 in HCC Tissues and CellsThe expression of Eph B3 in liver cancer and cell tissues was detected by RT-PCR and Western blot.The results showed that CRP was highly expressed in hepatocellular carcinoma cells,and the expression of Eph B3 and CRP were consistent in HCC.(2)Effect of Eph B3 on Migration and Invasion of Hepatoma CellsAfter transfection of hepatoma cells with Eph B3 s i RNA,transwell assay results showed that inhibition of Eph B3 expression in hepatoma cells can attenuate the ability of migration and invasion of hepatoma cells.4.CRP interacts with Eph B3 to regulate the MAPK/ERK pathway(1)Interaction between CRP and Eph B3Co-immunoprecipitation experiments were performed using CRP and Eph B3 as bait proteins,and the interaction between CRP and Eph B3 was verified by western blot.(2)Relationship between Eph B3 and MAPK/ERKCo-immunoprecipitation experiments using Eph B3 as bait protein confirmed that Eph B3 can precipitate MAPK and ERK.Confocal experiments showed that there is a co-expression region of Eph B3 with MAPK and ERK in hepatoma cells.After transfection of Eph B3 si RNA in hepatoma cells,the activation of MAPK/ERK pathway was significantly inhibited.(3)Effect of CRP on MAPK/ERK Pathway,the expression of HIF1α and MMP9Western blot analysis showed that the MAPK/ERK pathway,the expression of HIF1α and MMP9 were significantly changed after treatment of hepatoma cells with CRP antibody and or CRP recombinant protein.Conclusion:1.High level of CRP promotes the progression of liver cancer.2.There is an interaction between CRP and Eph B3.3.Activation of MAPK/ERK pathway after CRP binding to Eph B3 increased the expression of HIF1α in hepatocellular carcinoma cells and induced the increase of intracellular MMP-9 expression,which caused migration and invasion of liver cancer cells. |
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