Comparative Analysis Of The Proliferation And Related Signaling Pathways Based On Proteomics Of Human Hepatoma Cells Treated By Three Multi-RTK Inhibitors

Primary liver cancer is a common malignant tumor in clinics.According to histopathology,it can be divided into three types:hepatocellular carcinoma（HCC）,intrahepatic cholangiocarcinoma（ICC）and HCC-ICC mixed type.The most prominent epidemiological feature of primary liver cancer is that the proportion of HCC in primary liver cancer is as high as 90%～95%.The Guidelines for the Diagnosis and Treatment of Primary Liver Cancer in China（2022 edition）indicats that the incidence rate of HCC is the fourth highest in the epidemiological survey of malignant tumors in China,especially the mortality rate of HCC is the second highest.In the world,HCC is also one of the malignant tumors that seriously threaten human health.The incidence rate and mortality of HCC showed an increasing trend every year.Liver resection or liver transplantation is very important in the treatment of HCC,but only 20%～40%of patients can obtain radical surgery.Many patients lost the opportunity of radical surgery at the time of initial treatment due to various reasons.Among the molecular targeted therapeutic agents,sorafenib,lenvatinib and regorafenib are the most used multi-receptor tyrosine kinase（multi-RTK）inhibitors in clinical practice,and play a very important role in the treatment of advanced HCC patients.Multi-RTK inhibitors can directly inhibit the proliferation of hepatoma cells,and induce their apoptosis in addition to their important roles in angiogenesis inhibition of tumor tissue and regulation in cellular immune response.However,the status of the three drugs in the clinical guidelines is slightly different.Sorafenib and lenvatinib are the first-line drugs for clinical treatment of advanced HCC.Previous studies have shown that compared with sorafenib,lenvatinib can prolong the progression free survival（PFS）and objective response rate（ORR）of patients with liver cancer,and is more suitable for Asians.Regorafenib is a second-line drug for the treatment of liver cancer,which is often used for sequential treatment after the failure of sorafenib first-line treatment,so that the survival of patients with advanced liver cancer can benefit significantly.The above differences all suggest that the mechanism of the three drugs may be different,especially the exact mechanism that leads to the difference in efficacy of the three drugs is still unclear.We have explored and studied this research hotspot.In this study,we first conducted a meta-analysis of the clinical efficacy of first-line molecular targeted drugs sorafenib and lenvatinib in the treatment of advanced HCC.To explore the exact mechanism behind the differences in efficacy of commonly used first-line and second-line multi-RTK inhibitors in clinical practice,we analyzed the effects of sorafenib,lenvatinib and regorafenib on the proliferation,cell cycle distribution and apoptosis of the human hepatoma Huh-7 cells.In order to further analyze the potential mechanism of the difference of sorafenib,lenvatinib and regorafenib on Huh-7cell cycle and apoptosis,we used the quantitative proteomics technology based on Tandem Mass Tag（TMT）to analyze and compare the different effects of sorafenib,lenvatinib and regorafenib on proteomics of Huh-7 cells at half-maximal inhibitory concentration(IC₅₀).Focusing on the shared differential proteins of the three drug group cells for signal pathway analysis,in order to understand which signal pathway proteins related to cell cycle,DNA synthesis and apoptosis changed when sorafenib,lenvatinib and regorafenib inhibited the proliferation of human hepatoma Huh-7 cells.In particular,whether the changes of these signal pathway proteins are significantly different among the three groups of sorafenib,lenvatinib and regorafenib,in order to provide important reference value for the individualized selection and application of commonly used multi-RTK inhibitors in clinical practice.PartⅠClinical efficacy of sorafenib and lenvatinib in the treatment of advanced hepatocellular carcinomaObjective:To compare the clinical efficacy of lenvatinib and sorafenib as first-line molecular targeted drugs in the treatment of advanced hepatocellular carcinoma.Methods:Through searching Cochrane,Web of Science,Pub Med,CNKI,Wanfang,and other databases,all the literatures on the comparison of the clinical efficacy of sorafenib and lenvatinib as the first-line molecular targeted drugs in the treatment of advanced hepatocellular carcinoma were collected.The search date was from January 1,2019 to December 31,2021.According to the established inclusion and exclusion criteria for literature,we conduct literature screening,data organization,and literature evaluation.Evaluation indicators include overall survival（OS）,progression-free survival（PFS）,and objective response rate（ORR）.The included literatures were subjected to meta-analysis using Stata 11.0（Stata Corporation,College Station,TX）software.Results:1.Basic characteristics and quality evaluation of literaturesAccording to the search strategy and manual search,355 articles were initially obtained.After screening,6 articles were finally included,with a total of 1465 patients,including 582 cases in the lenvatinib group and 883 cases in the sorafenib group.Overall,in the literatures,the clinical indicators of the two groups were well balanced.The proportion of male patients was relatively high,and virus infection was the main basis of liver disease.Most patients had a liver function grading of Child-Pugh A,and liver cancer staging was mainly Barcelona Stage C.2.Observational indicators for clinical meta-analysisA total of 4 articles reported ORR,2 articles reported PFS,and 3 articles reported OS data.Fixed effect model analysis was used.The results showed that compared with the sorafenib group,the lenvatinib group was better in ORR（OR=11.16,95%CI:6.08-20.48,P<0.001）,PFS（OR=0.49,95%CI:0.34-0.71,P<0.001）.Using a random effects model analysis,there was no statistically significant difference between the two groups of OS（OR=0.70,95%CI:0.39-1.24,P=0.220）.Brief summary:In the treatment of patients with advanced hepatocellular carcinoma,compared with sorafenib,lenvatinib can improve the ORR of patients and prolong the PFS of patients,but whether sorafenib or lenvatinib was selected,the OS of patients in the two groups had no difference.PartⅡ A comparative study on the effects of three multi-RTK inhibitors on cell cycle and apoptosis in hepatoma carcinoma cells Objective:To compare and analyze the effects of sorafenib,lenvatinib and regorafenib on the proliferation,cell cycle distribution and apoptosis of human hepatoma Huh-7 cells.Methods:In the cell proliferation experiment,Huh-7 cells were seeded in 96-well plates at 5×10³cells per well.Culture medium was changed to the respective sorafenib-,lenvatinib-and regorafenib-containing medium after the cells firmly attached to the surface.The experiment included blank control（normal saline）medium group,solvent control（DMSO）medium group,and medium group containing sorafenib,lenvatinib or regorafenib（multiple drug concentrations of each agent）.Six replicate wells were used for each of the drug concentrations（n=6）.After 24,48 or 72 h of incubation with each inhibitor,the IC₅₀value of each drug inhibiting cell proliferation was determined.In cell cycle experiment,there were five groups,including blank control（normal saline）medium group,solvent control（DMSO）medium group,and medium groups containing sorafenib,lenvatinib or regorafenib(IC₅₀concentration of each agent).Three replicate wells were used for each of the drug（n=3）.After 72 h of incubation with each inhibitor,the cell samples were collected and detected by Coulter EPICS XL flow cytometer with EXPO32-ADC software,to obtain the information about cell distribution in G₀/G₁,S and G₂/M phases.In apoptosis experiment,the experimental procedures including the cell culture condition,experimental groups and drug-incubation were the same as the cell cycle experiment.After 72 h of incubation with each inhibitor,the cell samples were collected and detected by Coulter EPICS XL flow cytometer with EXPO32-ADC software,to obtain the information about early apoptosis,late apoptosis,necrotic cells,and normal cells.Results:1.Compare the effects of three multi-RTK inhibitors on Huh-7 cell growth curvesSorafenib,lenvatinib and regorafenib inhibited the proliferation of Huh-7cells in a concentration-and time-dependent manner,respectively.After the Huh-7 cells were incubated with multi-RTK inhibitor for 72 h,the IC₅₀value of sorafenib,lenvatinib or regorafenib was 3.738（3.504-3.987）μM,0.814（0.648-1.023）μM or 1.322（1.215-1.437）μM.The IC₅₀value of lenvatinib was significantly lower than that of regorafenib（P<0.01）,and the IC₅₀value of regorafenib was significantly lower than that of sorafenib（P<0.01）.After Huh-7 cells were incubated with the three inhibitors for 72 h,the maximum inhibition rates of sorafenib（21μM）,lenvatinib（35μM）and regorafenib（21μM）on Huh-7 cell proliferation reached 99.8±0.2%,83.4±0.5%and 98.7±0.1%,respectively.2.Compare the effects of three multi-RTK inhibitors on cell cycle of Huh-7cellsA significantly higher percentage of cells in G₀/G₁phase（65.4%）and a significantly lower percentage of cells in S phase（23.3%）in IC₅₀sorafenib group,comparing to the G₀/G₁phase（49.6%）and S phase（36.3%）in DMSO group（P<0.01）.Lenvatinib and regorafenib had similar effects to sorafenib on G₀/G₁phase cells and S phase cells,but their effects were significantly stronger than sorafenib,respectively（P<0.05 and P<0.01）.In comparison with DMSO group,either lenvatinib or regorafenib at IC₅₀significantly deceased the percentage of cells in G₂/M phase（P<0.01）,but the inhibitory effect of sorafenib was not significant（P>0.05）.Meanwhile,the inhibitory effect on G₂/M phase of regorafenib was significantly stronger than that of sorafenib（P<0.05）.3.Compare the effects of three multi-RTK inhibitors on Huh-7 cell apoptosisDMSO did not induce any significant changes of Huh-7 cell populations in the four quadrants when compared with the control（P>0.05）.Sorafenib at IC₅₀significantly increased only the proportion of early apoptotic cells from6.7%of DMSO group to 11.1%（P<0.01）.Moreover,the early apoptotic cells,late apoptotic cells and necrotic cells in lenvatinib group were increased significantly by 1.3,121 and 64 times when compared with that in DMSO group,respectively（P<0.01）.Especially,regorafenib at IC₅₀significantly increased the late apoptotic cells and necrotic cells by 0.4 and 1.4 times when compared with lenvatinib（P<0.05 and P<0.01）.Brief summary:The present study established the satisfactory dose-response curves of the cell growth inhibition by sorafenib,lenvatinib and regorafenib in Huh-7 cells,and their IC₅₀values were 3.738μM,0.814μM and 1.322μM,respectively.Lenvatinib and regorafenib at IC₅₀might have a stronger inhibitory effect on DNA synthesis of Huh-7 cells,resulting in more cells arrested at G₀/G₁phase with less cells at S phase and G₂/M phase.The ability of lenvatinib to induce early apoptosis of Huh-7 cells was stronger than that of sorafenib and regorafenib,while the proportion of necrotic cells induced by regorafenib was2.4 times as large as that induced by lenvatinib.PartⅢ A comparative study on the proteomic effects of three multi-RTK inhibitors in hepatoma carcinoma cells Objective:To analyze proteomic changes in human hepatoma Huh-7 cells treated with sorafenib,lenvatinib and regorafenib at respective IC₅₀using quantitative proteomics technology based on tandem mass tag（TMT）.Methods:The human hepatoma Huh-7 cells were seeded as monolayer in 75-cm²culture flasks at 1.2×10⁶cells per flask and cultured in DMEM containing10%FBS,100μg/ml streptomycin,100 units/ml penicillin and 4.5 g/L glucose.There were five experimental groups,including blank control（normal saline）group,solvent control（DMSO）group,sorafenib IC₅₀group,lenvatinib IC₅₀group and regorafenib IC₅₀group.The experiment was repeated 3 times（n=3）.After the cells were incubated with sorafenib,lenvatinib or regorafenib for 72 h,the Huh-7 cells were digested and collected,and the total protein samples of Huh-7 cells were extracted.We obtained three batches of 33 samples（each batch consisted of 11samples）after finishing the following experimental steps:determination of protein concentration of the protein samples,SDS-PAGE electrophoresis analysis of the protein samples,protein reduction and protein alkylation reaction of the protein samples,enzymolysis and desalination of the protein samples,isotope labeling of the peptide samples,TMT-labeled peptide samples mixed to form 3 batches of samples,desalination of the 3 batches of samples,and component collection of the 3 batches of samples by RP-HPLC system.The TMT-labeled peptides were separated by a Thermo Easy-n LC 1000HPLC system coupled with an Orbitrap Fusion Mass Spectrometer using a Nano Flex source（Thermo Scientific,United States）.Proteome Discoverer 2.1software（Thermo Scientific,USA）,SEQUEST HT search engine and human proteome database（www.uniprot.org,updated in 2021.7）were used to search and analyze the data on the raw spectrograms.Principal component analysis（PCA）,hierarchical cluster analysis（HCA）,Venn diagram analysis and volcanic plot analysis were conducted in’Wu Kong’platform.Results:1.Quality control of proteomic investigation and quantitative detection of proteinsIn the five experimental groups,the protein concentration of three samples within each group did not show a significant difference from each other（all values within the theoretical range）,indicating that the protein extraction of each sample was qualified.The pattern of protein bands appeared on SDS-PAGE gel was clearcut without pollution,indicating the high quality of the extracted protein.A total of three batches（33 samples）were detected,including 18546 proteins in the first batch,17301 proteins in the second batch and 15901 proteins in the third batch.There were 1157 proteins as credible proteins,which were detected in the three batches.2.Principal component analysis of the total identified proteinsThe results of principal component analysis（PCA）showed that the proteins identified in nine samples of sorafenib,lenvatinib and regorafenib groups were significantly different between groups.In the PC1 direction,the nine samples in sorafenib,lenvatinib and regorafenib groups were significantly separated from the six samples in the control group and DMSO group.Meanwhile,the three samples within group of sorafenib,lenvatinib or regorafenib were overlapped.In addition,the samples in 0.1‰DMSO group and control group were almost completely overlapped.In the PC2 direction,the six samples in sorafenib and regorafenib groups were significantly separated from the three samples of lenvatinib group,but the the samples in 0.1‰DMSO group and control group were overlapped almost completely.3.Hierarchicalcluster analysis of the total identified proteinsThe horizontal direction in HCA analysis represented the distance or dissimilarity between proteins or clusters.Each sample in the cluster 1 and cluster 2 contained 6,521 proteins and 5,056 proteins respectively.In general,most of the proteins（6521）in cluster 1 obtained from sorafenib,lenvatinib and regorafenib groups were obviously downregulated when compared with those in the control group and DMSO group.On the contrary,most of the proteins（5056）in cluster 2 obtained from sorafenib,lenvatinib and regorafenib groups were obviously upregulated when compared with those in the control group and DMSO group.Like PCA results,the samples in sorafenib,lenvatinib or regorafenib group were clustered together within each individual group,and all of them were clearly separated from those in the control group and DMSO group.4.Volcanic plot analysis of differential proteins between sorafenib and DMSO groupsThe results of volcanic plot analysis showed 1165 differential proteins identified between sorafenib and DMSO groups in the Huh-7 cells incubated with IC₅₀sorafenib or DMSO for 72 h（P<0.05）including 565 upregulated proteins and 600 downregulated proteins.5.Volcanic plot analysis of differential proteins between lenvatinib and DMSO groupsThe results of volcanic plot analysis showed 2739 differential proteins identified between lenvatinib and DMSO groups in the Huh-7 cells incubated with IC₅₀lenvatinib or DMSO for 72 h（P<0.05）including 982 upregulated proteins and 1757 downregulated proteins.6.Volcanic plot analysis of differential proteins between regorafenib and DMSO groupsThe results of volcanic plot analysis showed 2010 differential proteins identified between regorafenib and DMSO groups in the Huh-7 cells incubated with IC₅₀regorafenib or DMSO for 72 h（P<0.05）including 855upregulated proteins and 1155 downregulated proteins.Brief summary:From the human hepatoma Huh-7 cells incubated with sorafenib,lenvatinib or regorafenib at IC₅₀for 72 h,11577 proteins were identified basing on TMT-labeled peptides analyzed by a Thermo Easy-n LC 1000 HPLC system coupled with an Orbitrap Fusion mass spectrometer,and the differentially expressed proteins of 1165,2739 and 2010 were found in the sorafenib,lenvatinib and regorafenib treatments,respectively.Among the three multi-RTK inhibitors,the effect of renvartinib on protein expression in Huh-7cells was the strongest while that of sorafenib was the weakest.In addition,the total number of downregulated proteins by regorafenib or lenvatinib was 1.4times or 1.8 times higher than that of upregulated proteins.PartⅣ Mechanism analysis of the difference among three multi-RTK inhibitors in inhibiting the proliferation of hepatocellular carcinoma cells and their clinical significance in HCC treatmentObjective:To analyze the changes in signal pathway proteins related to the cell cycle,DNA synthesis and apoptosis of human hepatoma Huh-7 cells treated with sorafenib,lenvatinib and regorafenib at IC₅₀.Especially,whether there were significant differences among those changes in signal pathway proteins commonly induced by sorafenib,lenvatinib and regorafenib at IC₅₀.Methods:The 565 upregulated proteins in 1165 differential proteins of sorafenib group,982 upregulated proteins in 2739 differential proteins of lenvatinib group and 855 upregulated proteins in 2010 differential proteins of regorafenib group were merged to form the Venn diagram,by which we calculated the total number of commonly upregulated proteins between the two groups of sorafenib and lenvatinib,of sorafenib and regorafenib,of lenvatinib and regorafenib as well as that among three groups of sorafenib,lenvatinib and regorafenib.The 600 downregulated proteins in 1165 differential proteins of sorafenib group,1757 downregulated proteins in 2739 differential proteins of lenvatinib group and 1155 downregulated proteins in 2010 differential proteins of regorafenib group were merged to form the Venn diagram,by which we calculated the total number of commonly downregulated proteins between the two groups of sorafenib and lenvatinib,of sorafenib and regorafenib,of lenvatinib and regorafenib as well as that among three groups of sorafenib,lenvatinib and regorafenib.Using the DAVID Bioinformatics Resources（NIAID/NIH）（https://david-d.ncifcrf.gov/summary.jsp）on the computer,we logged in to the Functional Annotation Tool Upload page and uploaded all the commonly upregulated or downregulated differential proteins in the Step 1 Box.Then,on the Annotation Summary Chart page to find the results showed that the signal pathways involved in these differential proteins and the degree of protein changes were statistically different among the three groups of sorafenib,lenvatinib and regorafenib.Finally,in order to explore the clinical application value of the results of this study in the diagnosis of HCC patients and the therapeutic effects of three multi-RTK inhibitors when applied alone or in combination with other treatments,a comparative analysis was conducted on the differential proteins that affect signal pathway changes in domestic and foreign clinical research literature.To clarify which differential proteins can provide important reference value for the diagnosis,treatment,and prognosis prediction of primary liver cancer patients,especially for the selection of commonly used multi-RTK inhibitors in clinical practiceResults:1.Upregulated differential proteins shared by the three multi RTK inhibitorsSorafenib and lenvatinib at IC₅₀produced 1222 upregulated differential proteins in Huh-7 cells,325 of which were shared by both sorafenib and lenvatinib.Sorafenib and regorafenib at IC₅₀produced 741 upregulated differential proteins in Huh-7 cells,389 of which were shared by both sorafenib and regorafenib.Lenvatinib and regorafenib at IC₅₀produced 1333upregulated differential proteins in Huh-7 cells,504 of which were shared by both sorafenib and regorafenib.In addition,259 upregulated differential proteins in Huh-7 cells were shared by sorafenib,lenvatinib and regorafenib.2.Downreguled differential proteins shared by the three multi RTK inhibitorsSorafenib and lenvatinib at IC₅₀produced 1858 downregulated differential proteins in Huh-7 cells,499 of which were shared by both sorafenib and lenvatinib.Sorafenib and regorafenib at IC₅₀produced 1278 downregulated differential proteins in Huh-7 cells,477 of which were shared by both sorafenib and regorafenib.Lenvatinib and regorafenib at IC₅₀produced 2047downregulated differential proteins in Huh-7 cells,865 of which were shared by both sorafenib and regorafenib.In addition,419 downregulated differential proteins in Huh-7 cells were shared by sorafenib,lenvatinib and regorafenib.3.KEGG pathway analysis of the commonly downregulated proteinsSorafenib,lenvatinib and regorafenib at IC₅₀commonly downregulated 419proteins in the Huh-7 cells treated by the three agents for 72 h,respectively（P<0.05）.Ten KEGG signal pathways were enriched for the 419 proteins significantly（P<0.05）,among which ranking of the top six KEGG pathways included spliceosome signaling pathway,DNA replication signaling pathway,cell cycle signaling pathway,m RNA surveillance signaling pathway,P53signaling pathway and nucleotide excision repair related signaling pathway.3.1 Cell cycle signaling pathwaySorafenib,lenvatinib and regorafenib at IC₅₀significantly decreased the PCNA expression（P<0.05）,and lenvatinib and regorafenib more significantly decreased the PCNA expression than sorafenib（P<0.05 and P<0.01）.Sorafenib,lenvatinib and regorafenib significantly decreased the expression levels of MCM3,MCM4,SKP1 and Bub3（P<0.05 and P<0.01）,respectively,and regorafenib more significantly decreased the MCM3expression than sorafenib（P<0.01）.Regorafenib reduced the expression levels of MCM3 and MCM4 by at least 28%in comparison with DMSO.Lenvatinib and regorafenib（but not sorafenib）significantly decreased the expression levels of cyclin B1 and Mad2,respectively（P<0.05）.In addition,sorafenib,lenvatinib and regorafenib significantly decreased the expression of Cdc20,and the effect of regorafenib was significantly stronger than that of sorafenib（P<0.05）.The expression level of Cdc20 in lenvatinib group and regorafenib group decreased to 50.4%and 46.8%of the level in DMSO group as well as 72.2%and 67%of the level in sorafenib group,respectively.Like the results of Cdc20 expression,the cyclin B1 expression level in lenvatinib and regorafenib groups decreased to 59%and 60.6%of the level in DMSO group as well as 80.4%and 82.5%of the level in sorafenib group,respectively.3.2 P53 signaling pathwaySorafenib,lenvatinib and regorafenib at IC₅₀significantly decreased the expression levels of BCL-x L,TSP-1,PAI-1 and p53 R2,respectively（P<0.01）.In addition,the downregulation in expression of BCL-x L and TSP-1 by lenvatinib and regorafenib was significantly stronger than that by sorafenib（P<0.05 and P<0.01）.The expression levels of TSP-1 and PAI-1 in lenvatinib group significantly decreased to 38%and 45.9%of the levels in DMSO group as well as 60.9%and 68.7%of the levels in sorafenib group,respectively（P<0.05）.3.3 Spliceosome signaling pathwayTwelve proteins downregulated significantly by the three multi RTK inhibitors were involved in the spliceosome signaling pathway.Sorafenib,lenvatinib and regorafenib at IC₅₀significantly decreased the expression levels of DDX5,DHX15 and e IF4A3（P<0.01）,and the effect of lenvatinib on the DHX15 expression was significantly stronger than that of sorafenib（P<0.05）.Among the five proteins of WBP11,BUD31,Prp19,SF3A1 and UAP56,sorafenib,lenvatinib and regorafenib significantly decreased the expression levels of four proteins except for SF3A1（P<0.05 and P<0.01）.In addition,the downregulation of BUD31 expression by lenvatinib or regorafenib was significantly stronger than that by sorafenib（P<0.01）.The expression level of SF3A1 in lenvatinib and regorafenib（but not sorafenib）groups was significantly decreased by more than 20%in comparison with that in DMSO group（P<0.05）.Among the four proteins of SPF30,Dib1,SART1 and SNRPF,sorafenib,lenvatinib and regorafenib significantly decreased the expression levels of three proteins except for SNRPF（P<0.05 and P<0.01）.Sorafenib and regorafenib（but not lenvatinib）significantly decreased the expression of SNRPF（P<0.05）.3.4 Signaling pathway involved in m RNA surveillanceSeven proteins（PAPOLA、PAPOLB、e IF4A3、UAP56、HBS1L、PABPC1and PABPC1L）downregulated significantly by sorafenib,lenvatinib and regorafenib at IC₅₀were involved in the m RNA surveillance signaling pathway.The three multi RTK inhibitors significantly decreased the expression levels of the 7 proteins（P<0.01）.There was no significant difference in the downregulation effect on each protein among the three multi RTK inhibitors.3.5 Signaling pathways involved in nucleotide excision repair and DNA replicationFour downregulated proteins（CUL4A,Polε4,RFC2 and PCNA）were related to mammalian nucleotide excision repair（NER）signaling pathway,and six downregulated proteins（PRIM1,Polε4.RFC2,PCNA,MCM3 and MCM4）were related to the DNA replication signaling pathway.Sorafenib,lenvatinib and regorafenib at IC₅₀significantly decreased the expression levels of CUL4A,Polε4,RFC2 and PCNA（P<0.05 and P<0.01）,and the effect of lenvatinib or regorafenib on the PCNA expression was significantly stronger than that of sorafenib（P<0.05 and P<0.01）.In DNA replication signaling pathway,the expression levels of PRIM1,Polε4,RFC2,PCNA,MCM3 and MCM4 were significantly downregulated by sorafenib,lenvatinib and regorafenib,respectively（P<0.01）,and the effects of regorafenib on the PRIM1,PCNA and MCM3 expressions and the effect of lenvatinib on the PCNA expression were significantly stronger than those by sorafenib（P<0.05 and P<0.01）.The PRIM1 expression level in regorafenib group significantly decreased to 61.9%of the level in DMSO group,77.8%of the level in sorafenib group and 83.6%of the level in lenvatinib group,respectively（P<0.01）.Brief summary:Downregulated 419 proteins were shared by sorafenib,lenvatinib and regorafenib at IC₅₀in Huh-7 cells.The ranking of the top six KEGG signaling pathways included spliceosome,DNA replication,cell cycle,m RNA surveillance,P53 and nucleotide excision repair,involving 33 downregulated differential proteins.Twelve proteins among them might be related to the machanisms underlying the stronger effects of lenvatinib and regorafenib on the reduction in number of cells in S phase,the cell cycle arrest at G₀/G₁phase and the induction of cell apoptosis in comparison with sorafenib.Among the 33 down regulated differentially expressed proteins,14 proteins can serve as reference indicators for evaluating and comparing the therapeutic effects of three multi-RTK inhibitors alone or in combination with other treatments,and predicting prognosis.4 proteins are expected to become potential reference indicators for guiding treatment and predicting prognosis.The other 15 proteins were first discovered in human liver cancer cells as signal molecule proteins closely related to the anti-proliferative effect of multi-RTK inhibitors on liver cancer cells;especially for the 3 proteins DHX15,PAPOLB,and HBS1L,their association with cancer has not been found domestically or internationally.Conclusions:1.In the treatment of patients with advanced hepatocellular carcinoma,compared with sorafenib,lenvatinib can improve the ORR of patients and prolong the PFS of patients,but whether sorafenib or lenvatinib was selected,the OS of patients in the two groups had not been prolonged.2.Lenvatinib and regorafenib might have a stronger inhibitory effect on DNA synthesis of hepatoma cells,resulting in more cells arrested at G₀/G₁phase with less cells at S phase and G₂/M phase.Compared with sorafenib,which can only induce early apoptosis with the weakest effect,lenvatinib and regorafenib can also induce late apoptosis and necrosis of hepatoma cells.And lenvatinib has the strongest ability to induce early apoptosis,while regorafenib has a stronger ability to induce necrosis and late apoptosis.3.The effect of renvartinib on protein expression in hepatoma cells was the strongest while that of sorafenib was the weakest.In addition,the total number of downregulated proteins by regorafenib or lenvatinib was higher than that of upregulated proteins.4.Downregulated proteins were shared by sorafenib,lenvatinib and regorafenib in hepatoma cells.The ranking of the top six KEGG signaling pathways included spliceosome,DNA replication,cell cycle,m RNA surveillance,P53 and nucleotide excision repair,involving 33 downregulated differential proteins.Twelve proteins among them might be related to the machanisms underlying the stronger effects of lenvatinib and regorafenib on the reduction in number of cells in S phase,the cell cycle arrest at G₀/G₁phase and the induction of cell apoptosis in comparison with sorafenib.5.It is recommended that for HCC patients,routine testing of the expression levels of 14 proteins（PCNA,MCM3,MCM4,Mad2,Cdc20,TSP-1,PAI-1,DDX5,SF3A1,e IF4A3,PABPC1,CUL4A,RFC2,and PRIM1）in cancer tissue or blood samples can evaluate and compare the therapeutic effects of three multi-RTK inhibitors alone or in combination with other treatments and predict prognosis;for HCC patients,exploratory testing of the expression levels of four proteins（p53R2,Prp19,UAP56,and SART1）in cancer tissue or blood samples should be conducted to evaluate whether they can become potential biomarkers of early HCC and potential indicators of efficacy and prognosis in patients receiving multi-RTK inhibitor therapy.The other 15 proteins are signaling molecules that we first discovered in human liver cancer cells and are closely related to the anti-proliferation effect of multi-RTK inhibitors on liver cancer cells;especially for the three proteins DHX15,PAPOLB,and HBS1L,their association with cancer has not been found domestically or internationally;It is urgent to carry out in-depth and detailed basic and clinical research.