Objective:The increase of tumor stiffness is one of the important risk factors for the malignant progression of tumor.The normal physiological structure of tumor tissue is gradually replaced by the disordered structure of tumor tissue preferentially.As a characteristic physical property of tissue,the stiffness will gradually increase with the progression of tumor.However,the significance of the physical characteristics of this abnormal tumor tissue in the process of tumor metastasis and the specific mechanism of regulating tumor metastasis remain unknown.Statistically,90%of tumor deaths are caused by metastasis.How to prevent tumor metastasis is also the main direction of scientific research.According to the current classical theory,tumor metastasis involves a series of continuous and dynamically related processes.In order to complete the metastasis cascade,tumor cells enhance their deformation capacity and tensile force during invasion and migration through morphological changes and dynamic flow of skeleton structure.Intermediate filaments coordinate tubulin and microfilaments to help regulate cellular mobility and cytoplasmic and nuclear synergies during metastasis.They also have flexible length variability and can be adapted to the biological needs of cells in different situations by forming longitudinal or transverse polymers.It remains to be further revealed how the increased stiffness in tumor tissues exerts a regulatory effect on intermediate filaments and through which specific regulatory mechanisms are involved.In this study,3D invasion experiment,live cell movement tracking system and western blot experiment were used to explore the role of stiffness in tumor cell invasion and movement by constructing three-dimensional model culture system with different stiffness,so as to evaluate the regulatory relationship between stiffness intermediate filaments.At the same time,matrix gelatin-collagen system was used to establish in situ injection models with different stiffness in vivo,so as to clarify the influence of stiffness on tumor metastasis in vivo model.Finally,the potential target drugs were analyzed by computer simulation docking system,and the effectiveness of the drugs was verified by in vitro animal experiments.Research methods:Part I:The influence of matrix stiffness on hepatocellular carcinoma metastasis1.Experimental materials:HCC cell line MHCC97H and HepG2,stiffness petri dish,nude mice2.Experimental methods:(1)3D invasion:The stiffness model was used as the MHCC97H and HepG2 cell culture system.Live cell dyes were used to stain the cells,and fluorescence observation was carried out by confocal fluorescence microscopy.(2)Cell track:MHCC97H and HepG2 cells were cultured in the stiffness model and photographed continuously for 10h.(3)In vivo metastasis:Matrix and collagen were used to construct an in vivo stiffness model,and highly metastatic cell line MHCC97H cells were injected in situ into the left lobe of mouse liver.(4)Tumor growth:Matrix and collagen were used to build an in vivo stiffness model,and MHCC97H cells were used for subcutaneous tumorigenosis.3.Experimental scheme:(1)Evaluate cell motion rate:A three-dimensional stiffness model(4kPa and 12kPa)was constructed.The 4kPa culture system was used as the control group and the 12kPa culture system as the experimental group.Real-time observation of the cells was carried out using the live cell real-time imaging system,and the cell coordinates were obtained by Image J.R language(version 4.2.3)for Chemotaxis and migration tool(CAMT)input file format,CAMT for multicellular motion trajectory tracking and visualization,and cell motion rate evaluation based on the data;(2)Evaluation of cell invasion ability:In the stiffness model culture,the 4kPa group was used as the control group and the 12kPa group as the experimental group.The cells were stained for live cells,and the three-dimensional culture system was computed by fluorescence confocal microscope and three-dimensional reconstruction was performed by NIS element to evaluate the cell invasion distance.(3)Evaluation of metastasis ability:A stiffness model was established in vivo.The low-stiffness group was used as the control group and the high-stiffness group was used as the experimental group.(4)Evaluation of tumour growth ability:The tumour growth model of mice was established.The low-stiffness group was used as the control group and the high-stiffness group was used as the experimental group.Part two:The relationship between matrix stiffness and intermediate filamentsactin skeleton recombination1.Experimental materials:MHCC97H and HepG2 cells,stiffness Petri dishes2.Experimental methods:(1)Bioinformatics analysis:GEO database was used to obtain liver cancer metastasis related data,and R language(4.2.3)was used for grouping,differential analysis and differential gene enrichment analysis of transcriptome data.(2)Confocal fluorescence experiment:intermediate filaments and F-actin fluorescence staining were performed on cells in culture systems with different stiffness to evaluate their distribution and expression characteristics.(3)Western blot:To evaluate the expression of intermediate filament protein and YAP protein.(4)PCR experiment:The mRNA expression of intermediate filaments was analyzed.(5)3D invasion experiment:The stiffness model was used as the MHCC97H and HepG2 cell culture system.Live cell dyes were used to stain the cells,and fluorescence observation was carried out by confocal fluorescence microscopy.(6)Cell tracking experiment:MHCC97H and HepG2 cells were cultured in the stiffness model and photographed continuously for 10h.3.Experimental scheme:(1)To analyze the key biological processes in HCC metastasis:The GEO data set was obtained by R language(version 4.2.3),and sample groups(high/with metastasis,low/no metastasis)were constructed based on phenotypic data.The low/no metastasis group was taken as the control group and the high/with metastasis group as the experimental group.The differential gene comparison between the experimental group and the control group was conducted using limma package(3.54.2).ggplot2(3.4.2)was used for image mapping,pheatmap(1.0.12)was used for heat mapping,and clusterProfiles(4.6.2)was used for differential gene enrichment analysis to identify key biological processes in metastasis.(2)Anisotropy evaluation:The matrix-collagen three-dimensional culture system was used to culture,with 4kPa as the control group and 12kPa as the experimental group.The distribution characteristics of F-actin were evaluated by confocal microscopy,and the Factin anisotropy was evaluated by Fribril.(3)Protein expression analysis:4kPa as the control group and 12kPa as the experimental group were used to evaluate the expression of intermediate filament protein(vimentin)by western blotting.(4)mRNA expression detection:qRT-PCR assay was used to evaluate the mRNA expression of intermediate filaments.(5)Lentivirus cell line construction:Lentivirus constructed intermediate silk protein vimentin knockdown/high expression cell lines,vimentin knockdown and overexpression cell lines were constructed,and transfection was analyzed by PCR and western blot.(6)Anisotropy analysis:confocal fluorescence was used to analyze the co-localization,expression and distribution characteristics of intermediate filaments and F-actin under different treatment factors,and Fribril was used to conduct anisotropy analysis according to the characteristics of F-actin.(7)Protein binding analysis:protein immunoprecipitation experiment was conducted to analyze the binding between intermediate filaments and F-actin.Part III:Batch screening of targeted intermediate filaments and preliminary validation in vivo1.Experimental materials:MHCC97H and HepG2 cells,stiffness Petri dish,nude mice2.Experimental methods:(1)Autodock vina docking:Protein structure files were obtained through PDB database,small molecule data were obtained by pubchem,and autodock vina was used for batch molecular docking.(2)3D invasion experiment:Stiffness model was used as MHCC97H and HepG2 cell culture system,cells were treated with benazepril in combination,cells were stained with live cell dyes,and cells were observed by fluorescence confocal microscopy.(3)Cell tracking experiment:MHCC97H and HepG2 cells were cultured in the stiffness model and photographed continuously for 10h.(4)In vivo metastasis experiment:Matrix and collagen were used to construct the in vivo stiffness model.The highly transferred cell line MHCC97H cells were injected in situ into the left lobe of mouse liver,and benazepril was injected intraperitoneally.3.Experimental scheme:(1)Batch screening of drugs:Python language(3.11.3)was used to obtain pubchem database data in batches,intermediate silk protein structure was obtained from PDB database,Pymol(server version)was used for batch pre-processing of molecules,Autodock vina was run on the server for batch docking,and text was sorted in batches on the server.Quantitative analysis of molecular-protein binding ability;(2)Evaluation of cell movement and invasion ability:In the stiffness model,12kPa group was used as the control to evaluate the effects of benazepril on invasion and movement ability of hepatocellular carcinoma cells by 3D invasion experiment and live cell tracking experiment.(3)Evaluation of metastasis ability:The stiffness culture system was constructed by matrix and collagen to determine the number of nodules in the liver and lung tissues of mice in the high-stiffness system in the saline group,verteporfin(VP)group,benazepril group,LPA group,VP+benazepril group and LPA+benazepril group,and to evaluate the number of metastatic nodules to determine the metastasis situation.Results:Part I:The influence of matrix stiffness on HCC metastasisIn vitro:1.Compared with the 4kPa group,the motility of MHCC97H cells in the 12kPa stiffness culture system and HepG2 cells in the 12kPa environment were enhanced through the live cell real-time monitoring system.2.Compared with 4kPa environment,3D invasion experiment found that the invasion ability of MHCC97H and HepG2 was enhanced under 12kPa environment.3.The matrix gel collagen model was established in vivo.Compared with the low stiffness group,the number of intrahepatic metastatic nodules and lung metastatic nodules increased significantly in the high stiffness group.In vivo:1.The results of subcutaneous tumor formation showed that the tumor volume was significantly increased in the high-stiffness group compared with the low-stiffness group,but there was no intrahepatic metastasis or lung metastasis in both groups.Part two:The relationship between matrix stiffness and intermediate filaments actin skeleton recombination1.The results of bioinformatics analysis suggested that,compared with the low metastasis group,the differential genes of tissue sequencing data in the high metastasis group were enriched in the actin binding set.2.The increase of matrix stiffness leads to the change of cell morphology.3.The increase in stiffness can promote the expression of intermediate filaments in hepatocellular carcinoma cells.The spatial distance between F-actin and intermediate filaments is close,and the anisotropy of F-actin is remodeled.4.In three-dimensional spheres,intermediate filaments increased significantly with the increase of matrix stiffness,and the colocalization(spatial proximity)of intermediate filaments and F-actin was enhanced(yellow fluorescence was green and red superposition),and the expression of intermediate filaments showed uniform distribution(no local distribution was observed).The expression of intermediate filaments in metastatic nodules was higher than that in non-metastatic nodules.5.YAP activation and YAP expression increased in MHCC97H cells at 12kPa compared with 4kPa.Compared with 4kPa environment,YAP entry ratio and YAP expression of HepG2 cells increased significantly under 12kPa environment.6.Immunofluorescence experiments showed that the ratio of YAP nuclei and fluorescence density of MHCC97H cells increased at 12kPa compared with 4kPa.Compared with 4kPa environment,YAP nucleus ratio and fluorescence density of HepG2 cells increased at 12kPa environment.7.Immunofluorescence F-actin staining showed that the expression of intermediate filaments in MHCC97H cells increased with the increase of stiffness,and the anisotropy of F-actin in the progenies increased significantly,while the anisotropy of F-actin in the non-progenies decreased significantly.In HepG2 cell lines,the expression of intermediate filaments increased with the increase of stiffness,and the anisotropy of Factin in the progenies increased significantly,while the anisotropy of F-actin in the nonprogenies decreased significantly.The YAP activator LPA could effectively increase the expression of intermediate filaments,while the YAP inhibitor VP significantly inhibited the expression of intermediate filaments.8.In MHCC97H cells,YAP agonist LPA combined with interfilamentous knockdown expression under 4kPa environment could significantly inhibit cell motility and invasion ability after interfilamentous knockdown compared with LPA alone treatment group.HepG2 cells also showed a significant decrease in cell motility and invasion ability after intermediate filament knock down.Compared with VP alone,overexpression of intermediate filaments promoted cell movement and invasion at 12kPa,while knockdown of intermediate filaments inhibited cell movement and invasion compared with LPA alone.Part III:Batch screening of targeted intermediate filaments and preliminary validation in vivo1.Autodock vina was used to perform batch molecular docking.According to the absolute value of binding energy,diazepam,lorazepam and Benazepril were the most likely to bind to intermediate filamins.2.In MHCC97H cell line and HepG2 cell line,benazepril can significantly inhibit cell invasion and movement ability;3.In a high-stiffness environment,compared with the group without benazepril injection,the number of intrahepatic metastasis nodular decreased significantly after Benazepril injection,while the inhibitory effect of benazepril was no different from that of verteporfin alone,while LPA could promote liver metastasis and lung metastasis,and benazepril could block the metastasis promoting effect of LPA.Conclusions:1.The increase of matrix stiffness leads to the increase of the number of metastasis nodules in liver and lung,and can promote the invasion of liver cancer cells and the motor ability.2.Matrix stiffness regulates intermediate filament-mediated skeleton recombination through YAP signal;3.Benazepril can inhibit stiffness-related cell movement,invasion and hepatoma cell metastasis. |