| Objective:Macrophages is one of the most important immune cells in the tumor microenvironment,M1 type macrophages or classical activated macrophages is a type of macrophages in the tumor microenvironment,M1 macrophages by secrete proinflammatory cytokines and chemokines,and full-time presenting antigen,participate in the immune response,play the function of immune surveillance.The mechanism of the interaction between tumor cells and immune cells in the tumor microenvironment is an important factor affecting the biological behavior of tumor cells,which in turn also affects the prognosis of tumor patients.The relationship between tumor cells and tumor microenvironment attracts more and more attention of researchers,in which chemokines mediate the interaction between tumor cells and tumor microenvironment becomes the research focus.Previous studies have confirmed that chemokine ligand 5(CCL5)is significantly associated with the prognosis of colorectal cancer(CRC),but the biological significance and related clinical significance of CCL 5.The preliminary experimental results of our group showed that M1 type macrophages can secrete tumor necrosis factor ligand superfamily member 9(TNFSF9),and with high frequency microsatellite instability colorectal cancer(MSI-H CRC)cell membrane surface of tumor necrosis factor receptor superfamily member 9(TNFRSF9),and then affect the biological behavior of MSI-H CRC cells.This topic proposes a hypothesis and experimentally confirms this hypothesis: M1 macrophages secrete TNFSF9 and bind to TNFRSF9 on the membrane surface of MSI-H CRC cells,which can promote the secretion of CCL 5 by MSI-H CRC cells,and CCL 5 positively feedback the chemotaxis of M1 macrophages to MSI-H CRC cells in the tumor microenvironment.The interactions between CCL 5,M1-type macrophages,and MSI-H CRC were initially explored,Its role is expected to provide new molecular targets for the treatment and prognosis of MSI-H CRC.Methods:1.The correlation between CCL5 and TNFESF9 was analyzed by the UALCAN database and GEPIA database;the TCGA database was used to compare the expression differences of CCL5 between MSI-HCRC and MSI-L / MSS CRC,and the correlation between CCL5 and M1 macrophages in colorectal cancer using the TIMER2.0 database.2.The results of ELISA experiments showed that two strains of MSI-H CRC cells expressed more CCL 5 in the indirect culture environment than M1 type macrophages and MSI-H CRC cells.The results of q PCR experiments showed that both MSI-H CRC cells showed higher CCL5 m RNA expression in the indirect culture environment than MSI-H CRC cells.3.Inverted fluorescence microscopy and q PCR experiments showed that transfected TNFRSF9 si RNA significantly reduced the TNFRSF9 gene expression in MSI-H CRC cells DLD-1 and HCT-15 cells(P <0.0001).4.The results of ELISA experiments showed that CCL 5 in the si TNFRSF9 group of two MSI-H CRC cells expressed less CCL 5 in the indirect culture environment than CCL 5 in the si NC group in the indirect co-culture environment.The q PCR experiment results showed that the CCL5 m RNA expression in the si TNFRSF9 group of two MSI-H CRC cells was lower in the indirect culture environment than that in the si NC group in the indirect co-culture environment.5.The results of both scratch assay and Transwell experiments showed that CCL5 could promote M1 type macrophage migration.6.Western blot The experimental results showed that,compared with the si NC group of MSI-H CRC cells in the indirect co-culture environment,the expression of the NF-κ B pathway-related protein P-P65 in the si TNFRSF9 group of MSI-H CRC cells was decreased in the indirect culture environment.Results:1.CCL 5 was highly associated with TNFESF9 in the UALCAN database and GEPIA database(Pearson correlation coefficient of 0.74);CCL 5 in MSI-H CRC than MSI-L / MSS CRC in the TCGA database(P <0.0001);CCL 5 with M1 macrophages in colorectal cancer in the TIMER2.0 database.2.The results of ELISA experiments showed that two strains of MSI-H CRC cells expressed more CCL 5 in the indirect culture environment than M1 type macrophages and MSI-H CRC cells.The results of q PCR experiments showed that both MSI-H CRC cells showed higher CCL5 m RNA expression in the indirect culture environment than MSI-H CRC cells.3.Inverted fluorescence microscopy and q PCR experiments showed that transfected TNFRSF9 si RNA significantly reduced the TNFRSF9 gene expression in MSI-H CRC cells DLD-1 and HCT-15 cells(P <0.0001).4.The results of ELISA experiments showed that CCL 5 in the si TNFRSF9 group of two MSI-H CRC cells expressed less CCL 5 in the indirect culture environment than CCL 5 in the si NC group in the indirect co-culture environment.The q PCR experiment results showed that the CCL5 m RNA expression in the si TNFRSF9 group of two MSI-H CRC cells was lower in the indirect culture environment than that in the si NC group in the indirect co-culture environment.5.The results of both scratch assay and Transwell experiments showed that CCL5 could promote M1 type macrophage migration.6.Western blot The experimental results showed that,compared with the si NC group of MSI-H CRC cells in the indirect co-culture environment,the expression of the NF-κ B pathway-related protein P-P65 in the si TNFRSF9 group of MSI-H CRC cells was decreased in the indirect culture environment.Conclusions:1.While M1 type macrophages can promote CCL 5 secretion by MSI-H CRC cells and knockdown TNFRSF9 gene expression in MSI-H CRC cells,CCL 5expression is decreased in the environment of M1 type macrophages indirectly co-cultured with MSI-HCRC cells.2.CCL 5 is chemotactic to M1-type macrophages.3.The binding of TNFSF9 secreted by M1 type macrophages to TNFRSF9 on the surface of MSI-H CRC cells may promote the secretion of CCL 5 by MSI-H CRC cells via the NF-κ B signaling pathway. |
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