| Objective:To investigate the difference in expression and cell number of CD4~+T cell hypervariant genes in the tumor immune microenvironment and the effect of Par3 in the CD4~+T cell-mediated GZMA-PARD3 pathway on progressive osteosarcoma,and it provides a new direction and ideas for the clinical new targeted therapy of patients with progressive osteosarcoma.Methods:(1)Download sample data GSE152048 for primary osteosarcoma,recurrent and metastatic osteosarcoma through GEO database,and build a basic model for bioinformatics analysis,After the data were reduced,dimensionality reduction screening analysis was carried out to select various cell subsets in osteosarcoma tumor tissue,and localize and screen out T lymphocytes.As T lymphocytes were used as research objects to identify key marker genes in CD4~+T lymphocytes and locate specific CD4~+T lymphocytes number differences.(2)CD4~+T lymphocytes were compared and analyzed for the expression of highly variable genes.(3)Perform cell chat assays on the data,change CD4~+cells to represent T lymphocytes in the study object,To find out the interaction between CD4~+T cells and various cell subsets in osteosarcoma tissue.(4)Tissue samples of primary osteosarcoma and progressive osteosarcoma were collected clinically,HE staining and Masson staining were performed to detect the difference in tissue components,and the relative difference in the expression of the target gene was found by immunohistochemistry.(5)In vitro cell culture,cell scratch assay,and cck-8 cytotoxicity test were performed on OS cells to verify the effect of changing the protein activity level of Par3 on the cellular capacity of OS cells.Results:(1)By performing single-cell sequencing analysis on sample data,the expression of the marker gene IL7R and hypervariant gene GNLY of CD4~+T lymphocytes in primary osteosarcoma is higher than that in progress osteosarcoma,and cell chat detection showed that the GZMA-PARD3 pathway between CD4~+T cells and osteoblasts in progressive osteosarcoma was highly active,and the he content of Par3 in primary osteosarcoma is also much higher than in progressive osteosarcoma;(2)According to the results of HE staining,primary osteosarcoma cells are spindle-shaped in cell morphology,with more cytoplasm,relatively small nuclei,and shallow staining.Progressive osteosarcoma cells have a longer spindle degree,less cytoplasmic content,a nucleoplasmic ratio close to 1:1,and deeper nuclear staining.(3)The results of this study Masson staining showed:In primary osteosarcoma tumor tissue,the relative value of muscle fibers and collagen fibers can be approximately equal to 1:1,and nuclear staining is blue,the proportion of muscle fibers in the tumor tissue specimen of progressive osteosarcoma is much smaller than that of collagen fibers,and the nucleus is obviously blue-black,and echoes the HE staining results.(4)In the immunohistochemical localization and relative quantitative results,it can be seen that the number of two high-expression genes IL7R and GNLY in CD4~+T lymphocytes in the primary osteosarcoma tumor tissue was higher than that of progressive osteosarcoma tumor tissue.the Par3 content in the T cell-mediated GZMA-PARD3 pathway was also higher than in progressive osteosarcoma,it is confirmed that Par3 is a tumor suppressor gene,it can suppress progressive osteosarcoma by high expression of GZMA-PARD3.(5)According to cell scratch experiments,it is clear that simvastatin and 17-βestradiol have different effects on the opposite mechanism of action of Par3 protein and thus on OS cell migration.(6)Through the cytotoxicity test results of cck-8,different concentrations of simvastatin have different degrees of killing effect on OS tumor cells,while 17-βestradiol has the effect of promoting tumor cell growth.Conclusion:(1)CD4~+T lymphocytes are more abundant in primary osteosarcoma tumor tissue than in progressive osteosarcoma tumor tissue.(2)Par3 in the CD4~+T cell-mediated GZMA-PARD3 active pathway has inhibitory effect on progressive osteosarcoma.(3)Simvastatin can activate the GZMA-PARD3 pathway by promoting the production of Par3,which inhibits and kills OS tumor cells. |
