Research of the regulation of macrophage M1 polarization and influence of cell migration and invasion by FASN expression in glioblastomas
Objective To investigate the effect of the expression of fatty acid synthase(FASN)in glioblastoma(GBM)cells on polarization of human monocyte macrophage U937.Methods Stable interference RNA control group(U87sh-con group and LN229sh-con group)and FASN knockout experimental group(U87sh-FASN group and LN229sh-FASN group)were obtained by lentivirus transfection of U87 and LN229 cell lines.The cell supernatant(GBM conditioned medium)of the above 4 groups was collected and co-cultured with the induced by PMA differentiated U937 macrophages(the cell supernatant after co-culture used as the macrophage conditioned medium),and then the macrophage conditioned medium of the 4 groups were co-cultured with U251 cells.Protein expressions of FASN in U87sh-con,U87sh-FASN,LN229sh-con,LN229sh-FASN groups and COX-2 and ARG1 after co-culture of GBM conditioned medium with U937 cells were detected by Western blot(WB)and(or)immunofluorescence(IF).The proliferation after FASN knockout in U87 and LN229 of cells was detected by CCK-8 assay.The expressions of mature macrophage marker CD68,M1 marker CD11c,COX-2 and M2 marker IL-10 and ARG1 in macrophages were detected by quantitative real-time fluorescent polymerase chain reaction(qRT-PCR)after co-culture of GBM conditioned medium with U937 cells in 4 groups.The effect of macrophage conditioned medium on the migration and invasion ability of U251 cells was detected by Transwell assay.Results We successfully constructed U87 and LN229 cell lines with stable knockdown of FASN protein.WB and IF results showed that compared with sh-con group of corresponding cells,FASN protein expression in sh-FASN group of U87 and LN229 cells was decreased,and the differences were statistically significant(both P＜0.05).The CCK-8 results showed that at 48 hours,the difference of cell viability between U87sh-con and U87sh-FASN groups was statistically significant(588.391±10.032％vs.547.009±1.860％,P=0.002).At 24 hours and 48 hours,the differences of the cell viability between LN229sh-con and LN229sh-FASN groups were statistically significant(24 hours:235.033±1.598％vs.218.026±3.593％;48 hours:441.190± 5.196％vs.388.306±9.887％,both P＜0.05).There was no significant difference in cell viability between sh-con group and sh-FASN group in U87 and LN229 cells at 8 or 12 hours(all P＞0.05).The qRT-PCR results showed that compared with U937 cells,the relative expression of CD68 in U937 cells induced by differentiation and those co-cultured with four groups of GBM conditioned medium increased,with statistical significance(all P＜0.05).Compared with the sh-con group of corresponding cells,the relative expression levels of CD11c and COX-2 in the sh-FASN group of U87 and LN229 cells were increased,and the differences were statistically significant(all P＜0.05).However,there was no statistically significant difference in the relative expression levels of IL-10 or ARG1(all P＞0.05).The WB results showed that compared with the sh-con group of the corresponding cells,the expression of COX-2 protein in the sh-FASN group of U87 and LN229 cells was increased,and the differences were statistically significant(both P＜0.05),while there was no statistically significant difference in the expression of ARG1 protein(both P＞0.05).The results of transwell migration and invasion experiments showed that compared with the sh-con group of corresponding cells,the number of transmembrane penetrating cells in the U87,LN229 cell sh-FASN macrophage conditioned medium and U251 cell co-culture group decreased,and the differences were statistically significant(all P＜0.05).Conclusion Down-regulation of FASN expression in GBM cells can promote M1 polarization of macrophages,thereby inhibiting the migration and invasion ability of GBM cells.
GlioblastomaFatty acid synthasesMacrophagesM1 type polarizationCell migrationCell invasion
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