| The process of tumor development usually accompanied by a variety of physical forces,including solid stress,fluid shear force and extracellular matrix(ECM)stiffness and so on.These forces continue to act on cancer cells in tumor tissue and affect their behaviors.In recent years,the forces in tumor tissue,especially the matrix stiffness,has become important factors related to the occurrence and development of cancers.Hepatocellular carcinoma(HCC),as one of the most common solid tumors of human beings,is also characterized by increased stiffness of liver tissue.Studies have shown that extracellular matrix stiffness plays an important role in regulating the behaviors of hepatocellular carcinoma cells.However,little is known about the force transduction mechanism involved.Metabolic reprogramming is one of the main characteristics of cancer cells.Aerobic glycolysis(Warburg effect)is the main way of energy supply for cancer cells.As the two major characteristics of cancer,the effects of increased matrix stiffness and aerobic glycolysis on the biological behaviors of hepatocellular carcinoma cells have been proved by a large number of studies.In order to better understand the regulation of matrix stiffness on the biological behaviors of cancer cells,it is necessary to explore whether matrix stiffness has a regulatory effect on cancer cell metabolism.From the point of view of metabolism,an in-depth understanding of the mechanism of matrix stiffness regulating cell behaviors during the occurrence and development of HCC is of great significance for the development of new therapies for HCC.In this study,polyacrylamide hydrogels,which are usually used to simulate the stiffness changes of extracellular matrix in vitro,were used to prepare three kinds of matrix adhesives with different stiffness(65,25 and 54 k Pa)to simulate the stiffness of normal liver tissue,cirrhotic liver tissue and hepatocellular carcinoma tissue,respectively.Polyacrylamide hydrogels with different stiffness were coated with rat tail collagen Ⅰ and then were used to culture HCC cells for follow-up experiments.The effects of extracellular matrix stiffness on the migration and glycolysis metabolism of human HCC cells(Hep G2 and MHCC97L)and the related molecular mechanisms were investigated in order to provide theoretical support for understanding the mechanism of matrix stiffness on HCC cells migration from the metabolic point of view.The main experimental results are as follows:(1)Extracellular matrix stiffness regulates glycolysis metabolism and migration of HCC cellsHep G2 and MHCC97 L cells were inoculated on polyacrylamide hydrogels with three kinds of stiffness.48 hours later,the effects of matrix stiffness on the migration ability of Hep G2 and MHCC97 L cells were detected by scratch test and transwell test,respectively.The results showed that the migration ability of the two kinds of HCC cells increased with the increase of matrix stiffness.The gene level and protein expression of glycolysis-related enzymes were detected by q RT-PCR and western bolt.The results showed that with the increase of matrix stiffness,the expression of glucose transporter 1(Glut1),hexokinase Ⅱ(HK Ⅱ)and lactate dehydrogenase A(LDHA)increased significantly in the two kinds of HCC cells.And glucose consumption and lactic acid production also increased with the increase of matrix stiffness.Therefore,matrix stiffness can regulate the glycolysis metabolism and migration ability of HCC cells.After two specific si RNA were used to knock down HK Ⅱ in Hep G2 and MHCC97 L cells,the results of transwell assay showed that the migration ability of HCC cells on stiffer matrix was significantly inhibited and returned to the similar level as 6 k Pa.The above results suggest that the stiffness of extracellular matrix regulates the migration ability of hepatoma cells by affecting the glycolysis and metabolism of hepatoma cells.After two specific si RNA were used to knock down HK Ⅱ in Hep G2 and MHCC97 L,the results of transwell assay showed that the migration ability of the two kinds of HCC cells on the hard matrix was inhibited significantly difference in the migration ability of the two kinds of HCC cells in different matrix stiffness.(2)Matrix stiffness regulates glycolysis metabolism of HCC cells through YAP signalAs a mechanosensors and mechanotransducers,YAP can transform extracellular mechanical stimulation into intracellular biochemical signals to regulate the physiological and biochemical processes of cells.HCC cells Hep G2 and MHCC97 L were inoculated on polyacrylamide hydrogels with three kinds of stiffness.48 hours later,the expression of total YAP and phosphorylated YAP(p-YAP)was detected by western bolt.The results showed that the expression of total YAP increased and the value of p-YAP/ YAP decreased significantly with the increase of matrix stiffness.The results of immunofluorescence(IF)showed that YAP transferred from cytoplasm to nucleus with the increase of matrix stiffness.In addition,the expression of downstream target genes CYR61 and CTGF of YAP was detected by q RT-PCR.The results showed that the expression of CYR61 and CTGF increased significantly with the increase of matrix stiffness.These results suggested that the YAP activity of HCC cells increases with the increase of extracellular matrix stiffness.In order to determine the role of YAP in the regulation of matrix stiffness on glycolysis metabolism of HCC cells,two specific si RNA bands were used to knock down YAP in Hep G2 and MHCC97 L cells.After YAP knockout,the gene level and protein expression of glycolysis-related enzymes were detected by q RT-PCR and western bolt.The results showed that the gene and protein expression of glycolysis-related enzymes in the two kinds of HCC cells cultured on the stiffer matrix were significantly down-regulated,and the glucose consumption and lactic acid production of the two kinds of HCC cells cultured on the stiffer substrate were significantly lower than those in the control group.Therefore,extracellular matrix stiffness regulates glycolysis metabolism of HCC cells through YAP signal.(3)Matrix stiffness regulates YAP activity through JNK and p38 MAPK pathwaysHippo pathway is not the only pathway to regulate YAP.YAP may also be regulated by other intracellular signal pathways.Recent studies have shown that mechanical stimulation can regulate YAP activity through mitogen-activated protein kinase(MAPK)signal pathway.The expression of total extracellular signal-regulated kinase(ERK),c-jun N-terminal kinase(JNK),p38 MAPK and their phosphorylated levels in Hep G2 and MHCC97 L cells cultured on different matrix stiffness was detected by western bolt.The results showed that the level of p-ERK/ERK,p-JNK/JNK,p-p38/p38 increased with the increase of matrix stiffness.YAP expression was also detected by western bolt after treatment with ERK1/2,JNK or p38 MAPK pathway inhibitors.The results showed that the inhibitors of JNK and p38 MAPK pathway could significantly inhibit the expression of YAP in two kinds of HCC cells cultured on stiffer matrix,while the inhibitor of ERK1/2 pathway had no significant effect on the expression of YAP.The results of immunofluorescence showed that the fluorescence intensity of YAP in the stiffer matrix of JNK and p38 MAPK pathway inhibitor was significantly lower than that of the control group,while the treatment of ERK1/2pathway inhibitor had no significant effect on the expression of YAP.The expression of downstream target genes CYR61 and CTGF of YAP was detected by q RT-PCR after treatment with ERK1/2,JNK or p38 MAPK pathway inhibitors.The results showed that the expression of CYR61 and CTGF in the two kinds of HCC cells treated with JNK and p38 MAPK pathway inhibitor was significantly lower than that in the control group,but there was no significant difference between the ERK1/2 inhibitor group and the control group.These results suggested that extracellular matrix stiffness regulates YAP activity through JNK and p38 MAPK pathways.The migration of HCC cells on different matrix stiffness was detected after knockout YAP and treatment with JNK or p38 MAPK pathway inhibitors.The results showed that both knockout YAP,JNK or p38 MAPK pathway inhibitors significantly inhibited the migration of two kinds of HCC cells on stiffer matrix.It is suggested that YAP,JNK and p38 MAPK all play an important role in promoting the migration of HCC cells with the increase of matrix stiffness.Based on the above results,the increase of extracellular matrix stiffness may promote aerobic glycolysis and further promote the migration of HCC cells through MAPK-YAP-mediated mechantransduction.The results of this study provide an experimental basis for in-depth understanding of the role of extracellular matrix stiffness in the regulation of aerobic glycolysis metabolism and migration of HCC cells and its mechanical and biological mechanism,and provide theoretical guidance for the clinical treatment of HCC. |
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