The Molecular Mechanism Of Cell Geometry-induced Autophagy Of Human Melanoma Cells

Cells are embedded in a highly structured microenvironment in which the extracellular matrix(ECM)and neighboring cells impose specific boundaries that can affect not only the structural and mechanical properties of the cell,but also its polarity and function.Similarly,tumor cell development is inextricably linked to the microenvironment,which provides the metabolites and factors needed for the proliferation,spread,drug resistance,etc.behavior of tumor cells.The relationship between tumor cells and the microenvironment is both interdependent and mutually reinforcing,as well as antagonistic and combative.Micropatterning of cells is an efficient way to study cellular responses to specific microenvironmental cues,thus highlighting the critical role of the mechanical and geometric properties of the microenvironment in cell physiology.Autophagy is a highly conserved catabolic pathway that maintains homeostasis in the body and is responsible for the degradation of a large number of excess dysfunctional proteins and organelles.On the one hand,autophagy can produce a cytoprotective mechanism to remove its own aging proteins and damaged organelles;on the other hand,tumor cells can use the property of autophagy to reduce cell survival stress as a survival mechanism to resist chemotherapy and promote tumor cell growth and proliferation.However,the mechanism between tumor cell geometry and autophagy is not clear.Therefore,exploring the mechanisms of cell geometry and autophagy could provide a new way of thinking for clinical oncology treatment.Melanoma A375 cells were selected as subjects for this study.Different shapes of stamps were prepared by micro-contact printing technique to simulate the highly structured tumor microenvironment in vivo and to investigate the cell geometry.Mechanisms affecting autophagy of tumor cells.In this study,A375 were inoculated on triangular,circular,and circular cells with an area of 1000?m~2.Rectangular micropatterned substrates with aspect ratios of 1:1 and 1:4 were incubated for 24 h.Immunofluorescence technique was used to detect the amount of the cellular autophagy marker LC3B in different shapes of cells after treating by rapamycin.The number of cells LC3B was higher in ratio of 1:4 cells.The number of cellular LC3B puncta on the substrate was high.It was shown that YAP is a cellular mechanotransduction transcriptional co-activator.So YAP was further silenced to detect cell autophagy and immunofluorescence was used to detect mechanistic signals in different shaped cells nucleation of the sensitive molecule YAP,the experimental results show that after silencing YAP,the number of LC3B puncta decreases and the different shapes of the cells with different YAP nucleation profiles.By collecting nucleus pictures of different shaped cells by immunofluorescence technique and analyzing nucleus-related parameters by matlab,it is possible to the values of parameters such as transverse to vertical ratio and area of nucleus were found to be lower in cells with transverse to vertical ratio of 1:4.Immunofluorescence technique was used to detect the distribution of HDAC3 nuclei in cells of different shapes,and the results showed that HDAC3 had lower values in the transverse to vertical ratio.The level of nucleation was significantly reduced in cells with a ratio of 1:4,treatment with the myosin inhibitor Blebbistatin immunofluorescence was used to detect the distribution of nucleoplasm of HDAC3 in cells of different shapes,and the results showed that the entry of HDAC3 into the the nuclear level was significantly increased.Immediately afterwards,the expression of F-actin,myosin IIa was detected in different shapes of cells by immunofluorescence technique using after treatment with the myosin inhibitor Blebbistatin,the cellular stress fibers were detected by immunofluorescence technique.It was found that the expression level of F-actin,myosin IIa was significantly increased in cells with a transverse to vertical ratio of 1:4.In summary,in cells with a shape of 1:4 aspect ratio,the stronger cell contractility leads to lower HDAC3 nucleus levels and higher YAP nucleus entry levels,and higher autophagy levels.This shows that the cell geometry can affect the contractility of myosin,which in turn affects HDAC3 nuclear transport,which affects the nuclear level of the mechanically sensitive molecule YAP,and ultimately the level of autophagy.