The Effect Of SNF5 On The Biological Behavior Of Melanoma Cells In Response To Different Substrate Stiffness And Its Underlying Mechanism

Background:Malignant melanoma is one of the common and rapidly growing malignancies in clinical practice.The growth and development of melanoma depend on changes in the complex tumor microenvironment,the main components of which include cancer-associated fibroblasts,lymphocytes,myeloid suppressor cells,tumor-associated macrophages,dendritic cells,and extracellular components(Extracellular matrix,ECM),among which,changes in extracellular matrix ECM stiffness in the tumor microenvironment play an melanoma growth,metastasis and immunotherapy,but the underlying mechanisms and regulators of basal stiffness affecting melanoma development are unknown.The chromatin remodeling complex(SWItch/Sucrose Non-Fermentable,SWI/SNF)consists of several core and catalytic subunits,which function mainly by using energy generated from ATP(Adenosine-triphosphate)hydrolysis to alter the structure of nucleosomes and thus regulate gene transcription.SWI/SNF has been shown to be involved in regulating various aspects of cellular repair damage,transcriptional regulation,differentiation and immune response.Previous studies in our group found that the expression of BAF57(Brg-1-associated factor 57),a subunit of SWI/SNF,changes in response to cyclic stretch stimulation and further regulates the expression of cyclin D1 and other cell cycle proteins and variable splicing patterns of genes,suggesting that SWI/SNF can also respond to mechanistic signaling,but whether subunits of SWI/SNF respond to mechanistic signaling in melanoma and the specific regulatory mechanisms need to be further verified.Methods:1.To detect the effects of different stiffness substrate models on melanoma cell proliferation,EMT(Epithelial-mesenchymal transition)and immune escape.Firstly,3D micropillar structures with nominal height,diameter and spacing of 4μm were prepared,and different stiffness substrate models of 30:1 and 10:1 were prepared by adjusting the ratio of prepolymer and curing agent.The effects of different stiffness substrate models on cell proliferation were then examined by Ed U assay,followed by Q-PCR and Western blot to verify the effects of different stiffness substrate models on melanoma cell proliferation,EMT and immune escape related proteins.2.Screening and validation of SWI/SNF subunits that regulate the biological behavior of melanoma in response to mechanical signals.Firstly,we screened SWI/SNF subunits that respond to different stiffness changes by Q-PCR and Western blot assays;then we verified whether the effects of different stiffness substrate models on melanoma cell proliferation,EMT and immune escape behaviors require the participation of SWI/SNF subunits by sh RNA knockdown assays,Q-PCR and Western blot assays.3.To detect the regulatory effects of Sucrose Non-Fermenting gene number 5(SNF5)on A375 cells by establishing stable overexpression and stable knockdown cell lines.Firstly,SNF5 stable overexpression and stable knockdown cell lines were constructed using a lentiviral packaging system and examined at the gene and protein levels by Q-PCR and Western blot assays;then,the effects of SNF5 overexpression and knockdown on A375 cell proliferation were examined by Ed U assay and Western blot assay;meanwhile,the effects of SNF5 overexpression and knockdown on A375 cell proliferation were examined by The effects of SNF5 overexpression and knockdown on A375 cell proliferation were examined by Ed U assay and Western blot assay;meanwhile,the effects of SNF5 overexpression and knockdown on A375 cell migration,invasion and EMT-related protein expression were examined by Transwell assay and Western blot assay;finally,the effects of SNF5 overexpression and knockdown on A375cell immune escape related protein expression were examined by Western blot assay.4.To investigate the mechanism of SNF5 regulation of immune escape in melanoma cells.Firstly,the expression of STAT3/p-STAT3 on different hardness substrates was examined by Western blot assay;then the effect of different hardness substrates on the expression of STAT3/p-STAT3 was verified by sh RNA knockdown assay and Western blot assay.The effect of different stiffness substrates on STAT3/p-STAT3 required the involvement of SNF5;subsequently,the expression of STAT3/p-STAT3 in SNF5 stable overexpression cell lines and stable knockdown cell lines was examined by Western blot assays;finally,the expression of p-STAT3 in SNF5-regulated cell lines was verified by adding the inhibitor of p-STAT3-S3I201.STAT3 in the regulation of immune escape by SNF5 in melanoma cells.5.To analyze the effect of SNF5 on melanoma in the in vivo murine ruffled tumor assay and human melanoma tissue sample analysis assay.Firstly,we constructed a subcutaneous melanoma model in nude mice and a subcutaneous melanoma model in C57BL/6 mice to observe the effects of SNF5 knockdown on tumor weight,volume and survival of mice;then,after neutralizing CD8~+T cells in C57BL/6mice with CD8 antibody,we inoculated control cells and cells with stable knockdown of SNF5 under the skin of mice to observe the changes of tumor weight,volume and survival of mice.changes in survival period;subsequently,the infiltration of CD8~+T cells and the expression of PD-L1 in mouse tumor tissues were further analyzed by immunohistochemical assays;finally,the expression of SNF5 and PD-L1 in human nevus nigricans and human melanoma tissue samples were analyzed by immunohistochemical assays and correlation analysis was performed.Results:1.Harder substrates promote proliferation,EMT and immune escape of melanoma cells:Firstly,different hardness substrate models were prepared;then the expression levels of factors related to proliferation,EMT and immune escape were analyzed using Ed U,Q-PCR and Western blot assays,and the results showed that the percentage of Ed U-positive cells increased on harder substrates,while the expression of proliferation negative regulatory proteins The expression of p16 and p RB was suppressed;the harder substrate promoted the upregulated expression of EMT mesenchymal markers N-calmucin,VIM and transcription factors;the expression of immunosuppression-related factors PD-L1,PD-L2,FAS and IDO1 was upregulated on the harder substrate.2.SNF5,a core subunit of SWI/SNF,is an important factor for A375 response mechanistic signaling in melanoma cells:The subunit of SWI/SNF,SNF5,which is significantly up-regulated expression on stiffer matrix,was screened by Q-PCR and western blot;matrix stiffness with silencing SNF5 using sh RNA no longer down-regulated the expression of the proliferation-negative regulatory proteins p16 and p RB,no longer up-regulated the expression of N-cad,VIM and EMT transcription factors,and no longer up-regulated immunosuppression-related proteins PD-L1,FAS,PD-L2and IDO1 expression.3.SNF5 is important for regulating the proliferation,EMT and immune escape behaviors of melanoma cells A375:Overexpression of SNF5 using the lentiviral packaging system increased the percentage of Ed U-positive cells and suppressed the expression of the proliferation-negative regulatory proteins p16 and p RB;promoted the upregulation of the expression levels of the mesenchymal markers N-cad,VIM and the EMT transcription factors;and promoted the upregulation of the expression of the immunosuppression-related proteins PD-L1,FAS,PD-L2 and IDO1.Silencing of SNF5 using lentiviral packaging system reduced the percentage of Ed U-positive cells and promoted the expression of the proliferation-negative regulatory proteins p16 and p RB;downregulated the expression of the mesenchymal markers N-cad,VIM and the EMT transcription factors;and downregulated the expression of the immunosuppression-related proteins PD-L1,FAS,PD-L2 and IDO1.4.SNF5 regulates melanoma cell immune escape through STAT3/p-STAT3signaling pathway in response to mechanistic signaling:Phosphorylation levels of STAT3 were significantly increased on harder substrates;phosphorylation levels of STAT3 were not significantly changed after inoculation of SNF5 knockdown A375 cells on harder substrates.p-STAT3 phosphorylation was activated by SNF5 overexpression and inhibited by SNF5 knockdown.Inhibition of STAT3 phosphorylation with S3I-201inhibitor attenuated the effect of SNF5 overexpression on the expression of melanoma immune escape related factors.5.In vivo experiments showed that SNF5 knockdown down-regulated PD-L1expression and promoted CD8+T cell tumor microenvironment and inhibited tumor growth:In subcutaneous tumor loading experiments in nude mice without immunogenicity,SNF5 knockdown did not affect tumor growth and survival of mice;in subcutaneous tumor loading experiments in C57BL/6 mice with immunogenicity,SNF5knockdown slowed tumor growth and prolonged survival of mice;CD8 antibody neutralized the After CD8 antibody neutralized CD8~+T cells in C57BL/6 mice,SNF5knockdown no longer affected tumor growth and survival of mice;immunohistochemical results showed that SNF5 knockdown down-regulated PD-L1expression,promoted CD8~+T cell infiltration in the tumor microenvironment,and inhibited tumor growth.Both SNF5 and PD-L1 were upregulated in human melanoma tissue samples compared with benign nevi,and further correlation analysis showed a significant positive correlation between SNF5 and PD-L1 expression.Conclusion:In summary,this thesis analyzed the effects of different basal hardness on melanoma biological behaviors by constructing 3D topological structural mechanics models of different hardnesses in terms of cell proliferation,EMT and immune escape,and further determined the role and regulatory mechanism of SNF5 involved in melanoma development through activation of STAT3/p-STAT3 signaling pathway in response to mechanistic signals,and finally,in vivo load tumor The experiments clarified the mechanism by which knockdown of SNF5 inhibits tumor growth by affecting the infiltration of T cells in the tumor microenvironment.The findings of this thesis complement the role of mechanistic factors in melanoma development,and also provide new ideas for melanoma prevention and treatment.