The Effect Of BRAF Gene Silencing On Macrophage And Fibroblast Induced By Colon Cancer-derived Exosomes With BRAF^V600E Mutation

Part 1 Association of the mutation of BRAF^V600E with the tumor-associated macrophages and cancer-associated fibroblasts in colorectal cancerObjective: To investigate the relationship between BRAF^V600E gene mutation and tumor-associated macrophages,cancer-associated fibroblasts respectively in colorectal cancer.Methods:1.The clinical data and postoperative tumor tissue samples were collected from Aug.2014 to Mar.2019 in the Fourth Hospital of Hebei Medical University.10 patients with BRAF^V600E gene mutation in colorectal cancer and another 20 patients with BRAF wild-type colorectal cancer were used as controls to evaluate the relationship between BRAF^V600E mutation and clinicopathological parameters.2.Immunohistochemistry was used to stain postoperative tissue samples of 30 patients with colorectal cancer,the number of tumor-associated macrophages and cancer-associated fibroblasts counts was calculated to evaluate the relationship between tumor-associated macrophages,cancer-associated fibroblasts and BRAF^V600E gene mutation.Results:1.BRAF^V600E gene mutation in colorectal cancer was correlated to CA19-9 level and tumor differentiation degree（P<0.05）,but not associated with age,sex,site,TNM stage,lymph node metastasis,vascular cancer embolus,nerve invasion,PLT count,CEA level（P>0.05）;2.The number of M2 macrophages in BRAF^V600E mutant colorectal cancer tissue was significantly higher than that in BRAF wild type（P<0.05）;3.The expression of cancer-associated fibroblasts in BRAF^V600E mutant colorectal cancer tissue had no significant changes with BRAF wild type colorectal cancer tissues（P>0.05）.Conclusions:Patients with BRAF^V600E mutant CRC had higher preoperative CA19-9 levels and the tumor tissues were mostly poorly differentiated,and the number of M2 macrophages in tumor tissues was higher than that of BRAF wild type,inhibiting M2 macrophages infiltration into tumor microenvironment may be a strategy for treating BRAF^V600E mutant CRCPart 2 The effect of BRAF gene silencing on a macrophage and fibroblast induced by colon cancer-derived exosomes with BRAF^V600E mutationObjective: To investigate the effect of exosomes of BRAF^V600E mutant colon cancer cell line that silence BRAF gene on phenotype and secretion function of macrophage and fibroblast.Methods:1.Ultracentrifugation was adopted to separate exosomes from the HT29 cells supernatants or 1627 cells supernatants;The morphology and size of exosomes were detected by transmission electron microscope;The specific markers CD9 and CD63 of exosomes were detected by Western blot.2.Human acute monocytic leukemia cell line（THP-1）was induced into macrophages by PMA（100 ng/m L,48 h）.Then exosomes secreted from HT29 cells（HT29 exo group）or 1627 cells（1627 exo group）were co-cultured with THP-1 macrophage for 48 h,and the cells and supernatants were collected.Flow cytometry was used to detect the expression of CD163 in macrophage after co-culture,and ELISA was used to detect the levels of cytokines IL-6 and TGF-β1 in the supernatant of macrophage.3.Exosomes secreted from HT29 cells or 1627 cells were co-cultured with MRC-5 fibroblast for 48 h,and the cells and supernatants were collected.Western blot was used to detect the expression of α-SMA and FAP in fibroblast after co-culture,and ELISA was used to detect the levels of cytokines IL-6,TGF-β1 and VEGF in the supernatant of fibroblast.Results:1.The pellet harvested from HT29 and 1627 cell supernatants had a typical lipid bilayer membrane structure with membrane like structures sized in 30 to 150 nm under transmission electron microscope,which conformed to the morphological characteristics of exosomes,and all expressed exosomes specific markers CD9 and CD63;2.Effects of BRAF^V600E mutant colon cancer-derived exosomes on macrophage after silencing the BRAF gene（1）Compared with HT29 exo group,the expression of CD163 in macrophages in the 1627 exo group was decreased（P<0.05）;（2）Compared with HT29 exo group,the level of IL-6 secreted by macrophages was decreased and the level of TGF-β1 was increased in the 1627 exo group（P<0.05）;3.Effects of BRAF^V600E mutant colon cancer-derived exosomes on fibroblast after silencing the BRAF gene（1）Compared with HT29 exo group,the expression levels of α-SMA and FAP in fibroblast had no significant changes in the 1627 exo group（P>0.05）;（2）Compared with HT29 exo group,the level of IL-6,TGF-β1 and VEGF secreted by fibroblast was significantly decreased in the 1627 exo group（P <0.05）.Conclusions:After silencing BRAF gene,the exosomes of BRAF^V600E mutant colon cancer cell line can inhibit the differentiation of macrophage to M2 phenotype,and can also inhibit fibroblast from secreting IL-6,TGF-β1,and VEGF,gene therapy targeting BRAF may be the direction of future colorectal cancer treatment.