| Objectives: Glioma is the most common primary intracranial tumor in adults.Due to the biological characteristics of invasive growth,it is difficult to perform complete surgical resection.Even with the existing standard treatment,the prognosis of patients is still unsatisfactory.Therefore,there is an urgent need to find key targets for the invasion and progression of gliomas.Studies have shown that mi RNAs play an important role in the occurrence and development,cell proliferation,metastasis,invasion,angiogenesis and drug resistance of gliomas.And the role of Mi R-144-3p in the invasion and progression of various tumors has been confirmed.This study intends to explore the role of Mi R-144-3p/BCL6 axis in glioma invasion and progression.Methods: The expression differences of Mi R-144-3p and BCL6 in glioma and normal brain tissue were studied by q PCR and western blot technology.The RNA and protein expression of BCL6 in different tumors was analyzed by The Human Protein Atlas database and CCLE database.The relationship between BCL6 expression and prognosis of glioma patients was analyzed by CGGA database.The role of BCL6 in tumor immunity was analyzed by the TIMER database.The expression of BCL6-related genes and proteins was analyzed by STRING and GEPIA2 databases.The correlation between mi R-144-3p and immune cells in glioma was analyzed by TCGA database.Whether BCL6 is the target gene of Mi R-144-3p was verified by luciferase reporter system.After transfection of glioma cells with mi R-144-3p mimics,the changes of BCL6 expression in the cells were detected.CCK-8 experiments and transwell experiment were conducted to study the effect of up-regulated mi R-144-3p on the viability and cell invasion ability of glioma U251 cells.mi R-144-3p was overexpressed in U251 cells and co-transfected with BCL6 plasmid.Western blot,CCK-8 assay and Transwell assay were used to detect the changes of BCL6 expression,cell proliferation ability and cell invasion ability after co-transfection.Overexpressed mi R-144-3p or knocked out BCL6 overexpression to observe the changes in tumor size nude mice.Results: At the gene and protein levels,the expression of BCL6 in glioma was higher than that in normal brain tissue.By comparing the expression of BCL6 in different grades of gliomas by immunohistochemistry,it was found that BCL6 expression was higher in high-grade gliomas than in low-grade gliomas.Analysis of the CGGA database showed that the higher the expression of BCL6,the worse the prognosis of glioma patients.The TCGA database was analyzed by R software,and it was found that the top ten genes positively correlated with BCL6 were AC072022.1,NAV2,AKAP13,SRRM2,STAT5 B,AGO4,NUMA1,SAFB2,NHLRC2,PHF21A;the top ten negatively correlated genes were: GUK1,NDUFA4,MGST3,COX6A1,ATP5 MPL,FAM71E1,CLDN10,MAP1LC3 A,COX7A1,CDK5.The immune infiltration of tumor-associated fibroblasts in glioma was negatively correlated with the expression of BCL6 in tumor tissue by EPIC,XCELL,and MCPCOUNT algorithms.Based on the algorithm MCPCOUNTER,it was observed that BCL6 expression was positively correlated with the infiltration of monocytes and macrophages in relevant fibroblasts.Based on the TIDE and XCELL algorithms,it was observed that BCL6 expression was negatively correlated with the immune infiltration of M2 macrophages in tumor-associated fibroblasts.related.RT-q PCR data showed that the expression of mi R-144-3p was significantly decreased in glioma tissues,and the expression of BCL6 was significantly up-regulated in glioma tissues.Validation of the association between mi R-144-3p and BCL6 using the luciferase reporter gene method,U251 glioma cells co-transfected with mi R-144-3p mimic and WT-BCL6 3’-UTR compared with controls,luciferase activity was significantly down-regulated(P<0.001).However,in U251 cells transfected with MUT-BCL63’-UTR,the level of luciferase activity was not altered.After transfection of cells with mi R-144-3p mimic,the protein expression level of BCL6 was significantly down-regulated,whereas the protein expression level of BCL6 was enhanced after mi R-144-3p knockdown in U251 cells.Compared with the control group,CCK-8analysis showed that up-regulation of mi R-144-3p significantly decreased U251 cell viability,while down-regulation of mi R-144-3p significantly increased U251 cell viability(P<0.001).Transwell aasay showed that compared with the control group,the increased expression level of mi R-144-3p significantly inhibited the invasive ability of U251 cells,while the down-regulated expression of mi R-144-3p could enhance the invasive ability of U251 cells.In nude mice tumorigenesis experiments,we observed that compared with mice injected with mi R-NC or NC-sh RNA,respectively,the average tumors volume of mice injected with mi R-144-3p mimics or mice transfected with BCL6 sh RNA were significantly reduced.Conclusion: BCL6 is the target gene of mi R-144-3p in glioma cells.BCL6 plays an important role in mi R-144-3p-regulated glioma cell proliferation and invasion.Up-regulation of mi R-144-3p expression in cells or down-regulation of BCL6 expression in cells could inhibit tumor growth in nude mice.mi R-144-3p is expected to be one of the potential targets for glioma therapy. |
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