| Liver cancer is a malignant tumor with high morbidity and mortality which seriously endangers human life and health.Studies have shown that various types of cells in the tumor microenvironment,including host cells,macrophages,and endothelial cells,are involved in the occurrence and development of tumors.Recent students have found that cancer cells with stem cell properties in tumor play a non-negligible role in theoccurrence and development of tumors,and they are considered to be one of the most important reasons for clinical treatment failure,tumor recurrence,proliferation and poor prognosis.Studies have shown that a type of cell population with similar functions to normal stem cells,liver cancer stem cells(LCSCs),has also been found in liver cancer,which can induce the formation,renewal and proliferation of cancer cells,and is the "motive force" to maintain liver cancer.In HCC patients,resection of tumor lesions clears all HCC cells,but LCSCs remain vitality,resulting in the failure of clinical treatment,recurrence,and poor prognosis.Therefore,LCSCs are considered to be an important target cell subset for successful treatment of HCC.The regulation of the occurrence and development of liver cancer by LCSCs involves multiple factors,with the deeply study of the research,the regulatory role of mechanical factors in the tumor microenvironment in the process of tumor occurrence and development has received increasing attention.In the process of liver cancer,the biological behavior of LCSCs is affected by various forms of mechanical factors.Extracellular matrix stiffness,as an important mechanical feature of the liver tissue microenvironment,plays a key regulatory role in the occurrence and development of liver cancer.Therefore,elucidating the molecular mechanisms by the mechanical microenvironment of liver cancer regulates the proliferation,migration,differentiation and stemness maintenance of LCSCs is crucial for understanding the biological behavior of LCSCs and the regulation of cell fate,and is expected to provide new perspectives and new strategies of mechanical-biological insights for targeted therapy of liver cancer.Based on the above background,this study was based on mechanical microenvironment culture model of different substrate stiffness,to explore how substrate stiffness affect the biological behavior of LCSCs including proliferation and migration,and the deeply molecular mechanisms.Based on the molecular mechanism of substrate stiffness regulating LCSCs to screening small molecule drug targeting to LCSCs in the treatment of liver cancer,further to reveal its function role and molecular mechanism.The main contents and results of this research work are as follows:(1)Effects of substrate stiffness on the phenotype and the molecular mechanism of LCSCsUsing Hep3 B and HCCLM3 cell lines,LCSCs were successfully screened by in vitro sphere-forming culture method.Two kinds of polyvinyl alcohol(PVA)cell culture platform with different substrate stiffness,soft(Soft)and stiff(Stiff)were constructed by chemical cross-linking method to simulate the stiffness of normal liver tissue and advanced liver cancer respectively.The regulatory effects of different substrate stiffness on the phenotypes of LCSCs such as morphology,proliferation,migration and self-renewal ability were analyzed by q RT-PCR,Western blotting and immunofluorescence and other related cell/molecular biological techniques.Transcriptome sequencing technology was used to analyze the signaling pathway of substrate stiffness regulating to the LCSCs.Metabolomics technology was used to analyze the metabolic pathway of substrate stiffness regulating to the LCSCs,and related cellular/molecular biology experiments such as q RT-PCR,Western blotting and immunofluorescence were used to verify relevant molecular signaling pathways and metabolic pathways.In addition,in vivo tumorigenicity experiments in nude mice was used to exam the effect of substrate stiffness on the tumorigenicity of LCSCs.The results showed that soft substrate stiffness significantly promoted the proliferation,migration and colony formation of LCSCs,and was more conducive to the maintenance of stemness of LCSCs,LDHA is the rate-limiting enzyme of lactate,a metabolic end product of the glycolytic pathway,and LDHA is a key factor in the regulation of the phenotype of LCSCs mediated by matrix stiffness mechanics signaling.(2)Screening of anti-tumour drugs targeting LDHABased on the molecular mechanism of substrate stiffness regulating the LCSCs,the molecular docking technology was used to screen small molecule drugs targeting LDHA,and the antitumor effect of LCSCs was evaluated from the screened drugs.Based on the results of the molecular docking between LDHA and the drug,the target pathway of the drug and the cost of the drug,we finally screened out five drugs with lower minimum binding energy,lower cost,and different target pathways,including Cepharanthine(CEP),Diosmetin(DSM),Daphnoretin(DAP),Coptisine chloride(CPT)and Demethylzeylasteral(DML)were validated for targeting LDHA.The results showed that all the five drugs screened could significantly inhibit the malignant biological behavior of LCSCs,but the efficacy of DML was the best.DML can induce LCSCs cell cycle arrest in S phase,inhibit cell proliferation,and promote LCSCs cells Apoptosis,inhibited cell migration of LCSCs,and reduced the tumorigenicity of LCSCs in vivo.(3)DML targets LDHA to regulate the biological behavior of LCSCs through histone lactylationBased on the antitumor effect of DML targeting LDHA,we used q RT-PCR,Western blotting and other experiments to detect the expression of LDHA and genes and proteins related to cycle,apoptosis,migration and other related genes and proteins in LCSCs after treatment with DML.Correlation analysis verified the correlation between the expression level of LDHA and the expression of Ki67 and BCL2.The results showed that DML significantly inhibited the expression of LDHA in LCSCs,which was accompanied by a significant reduction of genes and proteins related to cycle,apoptosis,and migration.Subsequently,we used transcriptome sequencing technology and metabolomics to analyze the molecular mechanisms and signaling pathways of DML to LCSCs.The results showed that DML reduced the histone lactylation of LCSCs by inhibiting LDHA,the rate-limiting enzyme of a terminal metabolite of the glycolytic pathway-lactate,thereby eliminating the malignant biological behavior of LCSCs.DML had a significant inhibitory effect on the lactylation modification sites H3K9 la and H3K56 la on histones of LCSCs.Finally,we evaluated the antitumor properties of DML in vivo by using tumorigenicity experiments in nude mice.The results showed that in vivo,DML also reduced tumor volume by inhibiting the level of intraprotein lactate modification in tumor tissue.In conclusion,this study analyzed and detected the mechanical properties and biological phenotypes of LCSCs affected by stiffness of soft and stiff substrate by constructing PVA hydrogel substrate with different stiffness,and verified the effect of different substrate stiffness on the maintenance of stemness,proliferation,migration and other malignant tumor biological phenotypes of LCSCs,and clarified the relevant molecular mechanisms.Based on the molecular mechanism of substrate stiffness regulating the stemness of LCSCs,the drug screening of targeting LDHA to regulate histione lactylation modification was carried out through molecular docking,and the mechanism of the treatment of DML was revealed,proposed a new technology for targeting LCSCs to the treatment of liver cancer,provided a theoretical guidance for targeted intervention therapy of LCSCs in clinical applications. |
