Molecular Mechanism Of Albumin In Suppressing Invasion And Migration Of Hepatocellular Carcinoma

Background & Aims: Primary hepatocellular carcinoma(HCC)is the sixth leading malignancy worldwide and the third leading cause of cancer-related death,after lung cancer and colorectal cancer.The number of liver cancer patients in our country accounts for more than half of the world’s liver cancer patients,which is one of the countries with the heaviest burden of liver cancer.Despite significant advances in the diagnosis and treatment of HCC,the prognosis of HCC remains poor,with a 5-year survival rate of approximately 18%.Frequent intrahepatic and extrahepatic metastasis are major challenges to poor prognosis of HCC.In recent years,with great advance in the research on the molecular mechanism of tumgenesis,some targeted drugs such as ramvaritinib,regorafenib and capotinib have been developed for cancer treatment.However,the efficacy of these targeted agents in HCC is unsatisfactory due to the high genetic and epigenetic heterogeneity.Exploring the underlying mechanisms of HCC recurrence and metastasis and finding new therapeutic targets which have important clinical significance for improving the survival rate of HCC.Albumin,a member of the serum albumin family,is homologous to AFP sequence.It is a plasma protein with transport function in the body of birth offspring.The main functions are to maintain plasma colloid osmotic pressure,bind and transport,antioxidant,regulate inflammatory and immune responses,and protect vascular endothelial cells.A number of studies have shown that the low recurrence rate of HCC is associated with high serum albumin levels in patients,so the high serum ALB level is considered to be the main indicator of a good prognosis of HCC.And the literatures show that ALB can regulate the growth of HCC cells.However,the role and mechanism of ALB in HCC metastasis are still unclear.Therefore,in combination with previous studies,this study focused on ALB to explore the important role of ALB in the advancement of HCC and the mechanism of ALB affecting the migration and invasion of HCC,so as to provide a new strategy for the prevention and treatment of liver cancer.Methods: 1.Relationship between different serum ALB levels and clinical parameters in hepatocellular carcinoma patients Clinical records and relevant clinical data were retrospectively collected to clarify the prognostic role of ALB in HCC and its correlation with aggressiveness index.2.The effect of ALB on the migration and invasion of l hepatocellular carcinoma cells(1)Detection and expression of ALB in liver cancer tissues and cells Immunohistochemistry was used to detect the expression level of ALB in 50 cases of liver cancer and adjacent tissues,and the correlation analysis between the expression levels and clinicopathological characteristics was studied.Western blot analysis of ALB protein expression levels in different HCC cells and normal hepatocytes.(2)The effect of ALB on the migration and invasion of liver cancer cells After ALB si RNA interfered with Hep G2 and Huh7 cell lines respectively,the ability of hepatoma cells to migrate and invade was detected by scratch test and Transwell test.3.Screening of ALB-interacting proteins(1)Extraction of secreted proteins,i TRAQ technology combined with MS to screen ALB-interacting proteins Control si RNA was used as the negative control group,and ALB si RNA was used as the experimental group.Hep G2 cells were knocked down,the secreted proteins were concentrated and enriched.The differentially expressed proteins were identified by i TRAQ technology combined with mass spectrometry analysis.(2)Bioinformatics analysis Two online databases(PANTHER and Uniprot)were used to perform cluster GO analysis,KOG analysis and pathway analysis for these differentially expressed proteins.(3)ALB-interacting protein verification The differentially expressed proteins identified by bioinformatics analysis were verified by PCR and Western Blot.4.The effect of ALB-interacting proteins on the migration and invasion of liver cancer Hep G2 and Huh7 were knocked down by RPS3 A,NOTCH1 and u PAR si RNA,respectively,the ability of hepatoma cells to migrate and invade was detected by scratch test and Transwell test.5.ALB interacts with u PAR to regulate EMT and MMPs(1)After knockdown of ALB in Hep G2 and Huh7 cells,western blot analysis was performed to detect the protein expression levels of EMT markers,Vimentin,N-cadherin,E-cadherin and transcription factors Snail and Twist.(2)After knockdown of ALB in Hep G2 and Huh7 cells,western blot was used to detect the protein expression of MMP2 and MMP9,and gelatin zymography was used to detect their activity.(3)ELISA was used to verify the correlation between ALB and u PAR in the cohort population.6.Statistical analysis SPSS 26.0 was used for statistical analysis,and Graph Pad Prism(version 8.0)software was used for data analysis and graph display.p < 0.05 indicates statistical significance.All experiments should be conducted at least three times.Results: 1.Relationship between different serum ALB levels and clinical parameters in hepatocellular carcinoma patients ALB is not only an important prognostic factor in HCC patients,but also there is a negative correlation between serum albumin level and aggressiveness parameters of HCC.2.The effect of ALB on the migration and invasion of liver cancer cells(1)Detection and expression of ALB in liver cancer tissues and cells Immunohistochemical and Western blot analysis showed that ALB was low expressed in HCC tissues and cell lines.Clinicopathological baseline analysis indicated that the expression level of ALB was closely related to the correlation index of HCC invasiveness,and further found that ALB was associated with the metastatic potential of HCC cells.(2)The effect of ALB on the migration and invasion of liver cancer cells After ALB si RNA interfered with the Hep G2 and Huh7 cells,respectively,the scratch test and Transwell test indicated that ALB silencing promoted the migration and invasion of HCC cells in vitro.3.Screening of ALB-interacting proteins(1)Extraction of secreted proteins,i TRAQ technology combined with MS to screen ALB-interacting proteins 210 differentially expressed secretory proteins were identified by i TRAQ combined with mass spectrometry,180 of which were down-regulated and 30 up-regulated.(2)Bioinformatics analysis Clustering GO analysis of these differentially expressed proteins was performed by PANTHER and Uniprot,and the results indicated that the main biological processes involved included cellular process,metabolic process,biological regulation,stimulus response,multicellular organism process,localization and development process,etc.These proteins are mostly located in the cell part,organelle,extracellular region part,organelle part,membrane and membrane closure cavity.Molecular functions include binding,catalytic activity,molecular function regulation,and molecular transporter activity.KEGG pathway enrichment analysis showed that ALB interacting proteins were divided into 20 enrichment pathways.Complement and coagulation cascade is the most important pathway.4.The effect of ALB-interacting proteins on the migration and invasion of liver cancer After RPS3 A,NOTCH1,and u PAR si RNA knocked down Hep G2 and Huh7 HCC cells,respectively,scratch and Transwell assays showed that u PAR silencing significantly inhibited the migration and invasion of HCC.5.ALB interacts with u PAR to regulate EMT and MMPs(1)After knockdown of ALB in Hep G2 and Huh7 cells,western blot analysis was performed to detect the protein expression levels of EMT markers,Vimentin,N-cadherin,E-cadherin and transcription factors Snail and Twist.Western blot analysis showed that knockdown of ALB significantly increased the protein expression levels of Vimentin,N-cadherin,Snail and Twist,and decreased the protein expression of E-cadherin in Hep G2 and Huh7 cells.(2)After knockdown of ALB in Hep G2 and Huh7 cells,western blot was used to detect the protein expression of MMP2 and MMP9,and gelatin zymography was used to detect their activity.Western blot analysis showed that knockdown of ALB significantly increased the protein expression levels of MMP2 and MMP9 in Hep G2 and Huh7 cells.The activity of MMP2 and MMP9 was significantly increased by gelatinase spectrometry.(3)ELISA was used to verify the correlation between ALB and u PAR in the cohort population.There was a correlation between ALB and u PAR in the HCC group,and the Spearman correlation coefficient reached-0.6504(P<0.0001),showing a significant negative correlation.Conclusions: 1.Combined with the retrospective study of clinical cases,it can be seen that ALB is related to the aggressiveness of HCC.2.It has been confirmed that ALB is significantly down-regulated in liver cancer at both tissue and cell levels,and ALB can inhibit the migration and invasion of liver cancer cells.3.ALB may inhibit MMPs and EMT by regulating u PAR,thus inhibiting the migration and invasion of liver cancer.