| Background and significanceThe exact mechanism of cancer is not yet entirely clear, but it is known by a number of series of steps intrinsically linked composition, the interaction between tumor cells and host cells and the surrounding microenvironment, continuous, multi-stage extremely complex and dynamic process, with the abnormal deposition of extracellular matrix, as well as promote the development of tumor and its microenvironment of solid tumors mechanical properties occurs gradually been recognized, in a two-dimensional (2 dimensional,2D) and three-dimensional (3 dimensional,3D) environment Investigation report on the biological behavior of matrix mechanics of tumor cells affected more and more. The results show that matrix mechanics not only affect tumor cell morphology, proliferation, migration, invasion, will also affect the properties of cancer stem cells and tumor cell resistance and the like.Cell is a basic composition unit of organism. The vast majority of mammalian cell complex between components of extracellular matrix (extracellular matrixc, ECM), extracellular matrix (ECM), is made up of cells to synthesis and secretion of extracellular, distribution on the cell surface or molecules between cells, forming a complex macromolecular network, mainly some polysaccharide, protein or protein, is part of the animal tissue, do not belong to any of the cell. These substances constitute a complex space truss structure, it decided the characteristics of connective tissue, has the unique physical, biological, chemical, and biological and mechanical properties. Normal tissue is essential for organizations. ECM is not only itself can be used as receptors on cells in vitro, can also influence the spread of cytokines and can contact, and then adjust the cells and interaction of the feelings of microenvironment, which various signal cascade from the cell membrane to the nucleus. The physical properties of the ECM including stiffness matrix, porosity, solubility, space configuration and orientation (topology), and other features. Together these features determines the full features for its organization structure, and directly affect the behavior of the cell and its response to environment.Extracellular matrix having a connection, support and protect the role of water physics, compression and protection. Normal eukaryotic cells, in addition to mature blood cells, mostly to be adhered to in order to inhibit the apoptosis-specific extracellular matrix survive, given the dependence called (anchorage dependence). For example, once the epithelial cells and endothelial cells from the extracellular matrix programmed cell death occurs. Only certain cell adhesion to the substrate protein and RNA synthesis can only be carried out in a spread state DNA replication. Extracellular matrix may enhance or inhibit its activity by binding growth factor. Different effects of different extracellular matrix of cells. For example, fibroblast proliferation accelerate fibronectin matrix, proliferation slows in laminin matrix; and epithelial cell proliferation response to fibronectin and laminin opposite. Proliferation of tumor cells lost the set with dependency, it can proliferate in the semi suspension. In vitro experiments show that various cell out of nearly spherical shape when the extracellular matrix was a single free state. When a cell adhesion in different cells with the extracellular matrix can exhibit completely different shape. With a particular role in the extracellular matrix components and cell differentiation occurs through. For example, myoblast proliferation and maintain an undifferentiated phenotype on fibronectin; on laminin stop proliferation, differentiation, integration muscular tube. Speed and direction of the extracellular matrix of cell migration can be controlled, and provides a "scaffolding" for the cell migration. For example, fibronectin can promote the migration of fibroblasts and epithelial cells; laminin promotes migration of a variety of tumor cells. Haptotactic chemotaxis and migration of cells are dependent on the extracellular matrix. This is important in embryonic development and wound healing. Migration of cells depends on the assembly of cell adhesion and cytoskeleton. Induction of cell adhesion to the extracellular matrix when some form of focal adhesions, focal adhesion is contact between extracellular matrix and the cytoskeleton, "rivets."Degradation of ECM components, crosslinking, and reconstruction is a dynamic process, abnormal metabolism of ECM is often accompanied by the occurrence of diseases, studies have shown that in the real frequently during the occurrence and development of malignant tumor with ECM abnormal deposition, the changes of cell surface receptor expression and tissue stiffness increased. Using the imaging detection of early detection and diagnosis of the tumor tissue density or stiffness has important clinical significance. Abnormalities of the ECM on the one hand directly promote cell malignant transformation and transfer, on the other hand, through the influence of interstitial cells in the microenvironment, promoting tumor angiogenesis and inflammation, formed into a tumor microenvironment.A correct understanding of the mechanical characteristics of the tumor cells and the microenvironment extracellular matrix mechanics for the study is extremely important for tumor development and biological behavior of rats. With the abnormal deposition of extracellular matrix and promote the development of tumors and their microenvironment of solid tumors mechanical properties occurs gradually been recognized, reported that matrix mechanics study on the biological behavior of tumor cells affected more and more. The results show that matrix mechanics not only affect tumor cell morphology, proliferation, migration, invasion, will also affect the properties of cancer stem cells and tumor cell resistance and the specific characteristic.Hardening of the tumor extracellular matrix organization not only promoted the tumorigenesis and development process, but also increases its resistance. Abnormal deposition of extracellular matrix drug-induced tolerance attributed to two main reasons:(1) reduce or inhibit the effectiveness of a drug (such as to limit the spread and the rate of drug out of the cell). Drug penetration is lower than the density of collagen tissue in the tumor tissue with extensive collagen network. (2) tumor cells to increase damage tolerance of the drug (e.g., reduced sensitivity to apoptosis). Extracellular matrix-mediated integrin signaling can inhibit chemotherapy-induced apoptosis response. Studies have reported that compared to the soft substrate (1kPa), hard substrate (12kPa) can inhibit apoptosis induced by cisplatin drugs NSCLC cells survived after drug action on soft substrate tumorigenic cancer cells than on a hard substrate surviving cancer stronger. However, non-small cell lung cancer cells in the extremely hard (about 106 kPa) substrate sensitivity to cisplatin drugs is on 5kPa substrate 2 times, suggesting that screening anticancer drugs, when physiological and pathological study of intracellular ECM mechanical factors can not be ignored.Hippo signal transduction pathway is a newly discovered in recent years a signal transduction pathway. Studies have shown that the Hippo signaling pathway is involved in regulation of organ development to the size of the key signaling pathways, this view were first discovered in drosophila, the Hippo signaling pathway belongs to curb growing signaling pathways, in the process of evolution is very conservative, multicellular animals in fruit flies, mice, mammals are Hippo signaling pathways. The Hippo signaling pathway is found in fruit flies regulating cell size, volume of main organs signaling pathways, flies the Hippo signaling pathway of member can find corresponding homologue in higher organisms. Hippo signal transduction pathways by promoting cell apoptosis and restricting the size of cell proliferation regulation of organ development, more and more evidence that the Hippo signal regulation may be closely related with human tumorigenesis.Physiological reaction by the proteins involved in various body and adjustment, and the protein expression and activity as well as by various levels, various regulation. Protein phosphorylation and to phosphorylation is an important form of protein active regulation, through phosphorylation or phosphorylation, change the activity of the protein. The phosphorylation research, proteomics has become gene expression, an important aspect in the study of enzyme kinetics. Under physiological conditions, there are large amounts of nucleus of protein phosphorylation modification.In mammals, the Hippo signaling pathway upstream of the membrane protein receptor feel the growth inhibition of extracellular signals, after a series of kinase phosphorylation of complex cascade, will eventually phosphorylation downstream effect factor Yes-associatedrelated protein (YAP). Yes-associated protein phosphorylation (YAP) and cytoskeleton protein interactions, stranded in the cytoplasm, could not enter the nucleus to exercise its transcription activation function.Yes-associated protein (YAP) as the Hippo signaling pathways downstream of effect factor has the transcriptional activation function, with transcriptional regulation factor in the nuclei TEAD, promote the downstream gene expression, so as to promote cell growth, inhibiting cell apoptosis gene transcription, such as survivin, cyclinE, etc. And a few other gene regulation, such as:Mst1/2, WW45, Lats1/2 and the Mob, the upstream regulation pathway genes of any change or YAP excess expression will cause excessive growth of cells.In mammalian cells, the Hippo signaling pathway through the phosphorylation and promote cytoplasmic transfer to suppress Yes-associated protein (YAP) and its homologue TAZ function. TEAD family of transcription factors is the influence of evolution on conservative Yes-associated protein protein (YAP) the key factor of the biological functions.YAP is a candidate oncogene, and several other factors on the Hippo pathway is a tumor suppressor. If the Hippo-YAP pathway dysfunction leads to cancer cell loss of contact inhibition (cancer cells without contact inhibition will spread more easily, tumor will quickly spread). In Hippo-YAP signal pathway, YAP as the downstream signaling pathway is highly conserved central role factor in mammals, YAP play a biological role in the process, can not be responsible for every step of the process, it can provide in favor of cell proliferation and inhibition of apoptosis microenvironment, increasing instability before cell-associated malignant transformation genome, raise the level of downstream target gene expression, enhance the ability of cells malignant transformation, YAP in cell growth, differentiation, apoptosis plays a role in maintaining that it tissues and organs in the role played by the stability, if YAP once dysfunction, there conducive to the normal cells into malignant cell transformation.Non-small cell lung cancer occurrence, development, invasion and metastasis are dependent on extracellular matrix deposition and crosslinking, interstitial stiffness increasing, the tumor cells to soften at the same time, eventually forming mechanical heterogeneity of tumor microenvironment. Matrix mechanics by influencing the tumor cell proliferation, migration and transfer, EMT, tumor stem cell properties and resistance to regulate and control the occurrence and metastasis of tumors, however, about the Yes-associated protein (YAP) and extracellular matrix protein involved in the growth of the mechanics of non-small cell lung cancer research, has not been seen at home and abroad. This study will focus on Yes-associated protein (YAP) by extracellular matrix mechanics regulating the growth of non-small cell lung cancer cells were studied.Objective:using different hardness of matrix and RNA interference technology specific inhibition YAP non-small cell lung cancer cell protein expression, protein YAP and extracellular matrix mechanics, regulating the growth of non-small cell lung cancer.Methods:acrylamide and double the amount of crosslinking agent, acrylamide produce different cultivation matrix hardness, using cell immunofluorescence and western blot of different hardness medium qualitative non-small cell lung cancer cell lines in the SPCA-1 YAP in intracellular localization and protein expression level of liposome in 2000 as transfection reagent, the specificity of silent siRNA YAP gene transfection into MTA1 gene expression in different hardness of matrix cell lines, western blot test interference effect, and real-time fluorescent quantitative PCR, western blot detection of CTGF, AREG, Survivin, Ki67 mRNA and protein expression level.Results:by adjusting the acrylamide and double the amount of crosslinking agent, acrylamide produce different hardness of fibronectin-coated PA gel. The cells cultured in 40 kpa substrate, YAP mainly located in the nucleus. The cells cultured in the 4 kpa substrate, YAP, mainly located in the cytoplasm and nucleus of YAP significantly less than 40 kpa cells cultured on base board, YAP protein expression on the hard matrix after incubation increased significantly (P<0.001). And p-YAP and LATS1 protein expression in hard matrix decreased significantly after the training. Synthetic siYAP could inhibit the SPCA-1 YAP protein expression in cells, the cells cultured on 40 kpa substrate in YAP gene silence, cell proliferation rate significantly decreased (P< 0.05). The cells cultured in the 4 kpa substrate in YAP gene silence, no significant differences in cell proliferation rate. In addition to normal cells, the expression of YAP SPCA-1 in 4 kpa cell proliferation rate relative to the substrate culture in 40 kpa substrate cultured cell proliferation rate significantly decreased (P< 0.05). The cells cultured in 40 kpa, YAP gene silence, CTGF, AREG, Survivin and Ki67 mRNA and protein expression level decreased obviously (P<0.05), the cells cultured in 4 kpa, YAP gene silence, CTGF, AREG, Survivin and no decrease of Ki67 mRNA and protein expression level. Normal cells, the expression of YAP SPCA-1 in 4 kpa substrate relative to the culture of cells in 40 cells cultured on kpa substrate CTGF, AREG, Survivin and Ki67 mRNA and protein expression level decreased significantly (P< 0.05).Conclusion:the extracellular matrix hardness changed the biological behavior of non-small cell lung cancer cells, Yes-associated proteins (YAP) through the extracellular matrix protein involved in mechanics to adjust the growth of non-small cell lung cancer, revealing the Hippo-YAP signaling pathways through specific microenvironment regulates the growth of non-small cell lung cancer. |
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