Regulation Of Breast Carcinoma Metastasis By NMHCⅡA Through Integrin α5 Pathway

Breast carcinoma is the most common female malignancy, metastasis is the leading cause of death. Therefore, understanding the mechanism of invasion and metastasis of breast cancer cells is significance for the further treatment.Non-muscle Myosin Ⅱ(NM Ⅱ) is an important component of the cytoskeleton, also participating in a variety of life activities. NMⅡ is a hexamer structure composed of two nonmuscle myosin heavy chain(NMHC), a couple of regulatory light chain(RLC) and essential light chain(ELC), Which are thought to play a role in regulating and stabilizng nonmuscle myosin heavy chain. Phosphorylation of heavy chain can regulate cell motility。 so probably the heavy chain is the key aspect of regulation of cell motility. So whether NMHCⅡ involved in regulating of cell motility get our attention. Previous study showed that, NMHCⅡ plays an important role in regulating migration and invasion in tumor cells. When silenced NMHCⅡ,migration and invasion of tumor cells were limited. However, the regulation mechanism of this phenomenon remains unclear.Integrins are important transmembrane adhesion molecules located on the cell membrane, as a integrin heterodimer composed by α subunits and β subunits.The specific transmembrane domain of integrin can be divided into the extracellular and the intracellular domain. The extracellular domain participates in the regulation of different signaling pathways by binding to different ligands, delivering the chemical signals and mechanical signals. The intracellular domain of integrin interacts with the cytoskeletal proteins, leading to rearrangement of cytoskeletal proteins, resulting in a series of biological behavior. As it reported that, when increased the expression of integrin accompanied by increasing the extent of malignancy and the ability of invasion in tumor cells, which are closely associated with the cytoskeleton and contractile force, which may be related to the MLC phosphorylation by α5β1,but it remains to be seen whether NMHC involved.Above all, we speculate that, in the initial stage of tumor metastasis, integrin may regulate NMHCⅡphosphorylation activation through NMLC, resulting in cytoskeleton rearrangement and contractile force, thus triggering a series of biological activity of tumor cells as migration, invasion and so on.Objectives: To investigate the effect of integrin α5 pathway on breast cancer cells through NMHCⅡAMethods: 1.Immunohistochemical staining method to observe expression of integrin α5, NMHCⅡA activated form(p-S1943) in breast tissue. 2.In breast cancer, the relationship of integrin α5 and NMHCⅡA activated form.(1) Lentivirus transfection silence integrin α5 in MDA-MB-231 cells, immunofluorescence and flow cytometry is applied to detect transfection efficiency.Western blot, flow cytometry and immunofluorescence staining are used to detect silencing efficiency.(2)After silencing integrinα5, then detect the expression levels of p-S1943、NMHCⅡA by western blot.(3) The effect of silencing integrin α5 on the biological behavior of tumor cells.(1)Application of CCK-8 kit to detect effect of silencing of integrin α5 on cell proliferation.(2)Detect cell cycle after silence of integrin α5 by flow cytometry.(3)Detect the change of cell migration of silence integrin α5 by cell wound scratch assay.(4)The transwell is used to detect the effect of integrin α5 on invasion of MDA-MB-231 cell(5)The effect of silencing integrin α5 on cell adhesion ability of MDA-MB-231 cell via adhesion assay.(6)The measurement area is used to detect the ability of spreading after silencing integrin α5.Results: 1. Immunohistochemical staining found that the expression in invasive ductal carcinoma of the integrin α5, p-S1943 were higher than fibroadenoma and breast hyperplasia. and between invasive ductal carcinoma and hyperplasia of groups was statistically significance(P<0.05).And integrin α5 and NMHCⅡA, p-S1943 were co-localization. 2. The relationship of Integrin α5 and NMHCⅡA its activated form(1) Stably transfected MDA-MB-231 cell lines by flow cytometry and fluorescence detect transfection efficiency were more than 90%. Western blot and immunofluorescence staining confirmed silencing efficiency, confirming the serial number for the 16381 lentivirus has the highest efficiency of gene silencing.(2) Western blot and immunofluorescence staining showed that after silencing integrin α5, NMHCⅡA, p-S1943 expression decreased, also the position expressed by the cytoplasm and cellmembrane into the membrane expression.(3)The biological behavior of tumor cells after integrin α5.(1)Flow cytometry cell growth cycle showed knockdown α5 integrin had no significant effect on the MDA-MB-231 cell proliferation and cell cycle.(2) The cell wound scratch assay showed the migration efficiency of silence group were lower than the control group.(3)Cell invasion assay results showed that silencing integrin α5, the number of cells through matrigel was significantly lower than non-transfected group and the no-load group, and between groups was statistically significant.(4)Cell adhesion experiments showed that silencing integrin α5, the capacity of cell adhesion decreased compared with non-transfected group and the no-load group, and between groups were statistically significant.(5) Immunofluorescence staining showed that silencing integrin α5, cell spreading area was significantly lower than non-transfected group load combinations. And between the groups was statistically significant.Conclusions: 1. Integrin α5 regulates NMHCⅡA in breast carcinoma. 2. Integrin α5 can regulate tumor cell invasion, migration, adhesion, spreading and other biological behaviors. 3.Integrin α5 impacts breast carcinoma through NMHCⅡA.